Uses of recombinant colony stimulating factor-1

ABSTRACT

A colony stimulating factor, CSF-1, is a lymphokine useful in overcoming the immunosuppression induced by chemotherapy or resulting from other causes. CSF-1 is obtained in usable amounts by recombinant methods, including cloning and expression of the murine and human DNA sequences encoding this protein.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of copending U.S. patent application Ser.No. 876,819, filed June 20, 1986 now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 821,068, filedJan. 21, 1986 (now abandoned), which is a continuation-in-part of U.S.patent application Ser. No. 756,814, filed July 18, 1985 (nowabandoned), which is a continuation-in-part of U.S. patent applicationSer. No. 744,924, filed June 14, 1985 (now abandoned), which is acontinuation-in-part of U.S. patent application Ser. No. 728,834, filedApr. 30, 1985 (now abandoned), which is a continuation-in-part of U.S.Ser. No. 698,359, filed Feb. 5, 1985 (now abandoned).

TECHNICAL FIELD

The present invention relates to the various uses of recombinantlyproduced human colony stimulating factor-1 (CSF-1).

BACKGROUND ART

The ability of certain factors produced in very low concentration in avariety of tissues to stimulate the growth and development of bonemarrow progenitor cells into granulocytes and/or macrophages has beenknown for nearly 15 years. The presence of such factors in sera, urinesamples, and tissue extracts from a number of species is demonstrableusing an in vitro assay which measures the stimulation of colonyformation by bone marrow cells plated in semi-solid culture medium.There is no known in vivo assay. Because these factors induce theformation of such colonies, the factors collectively have been calledColony Stimulating Factors (CSF).

More recently, it has been shown that there are at least four subclassesof human CSF proteins which can be defined according to the types ofcells found in the resultant colonies. One subclass, CSF-1 results incolonies containing macrophages predominantly. Other subclasses producecolonies which contain both neutrophilic granulocytes and macrophages;which contain exclusively neutrophilic granulocytes; and which containneutrophilic and eosinophilic granulocytes and macrophages.

There are murine factors analogous to the first three of the above humanCSFs. In addition, a murine factor called IL-3 induces colonies frommurine bone marrow cells which contain all these cell types plusmegakaryocytes, erythrocytes, and mast cells, in various combinations.These CSFs have been reviewed by Dexter, T. M., Nature (1984) 309:746,and Vadas, M. A., et al, J Immunol (1983) 130:793.

The invention herein is concerned with the recombinant production ofproteins which are members of the first of these subclasses, CSF-1. Thissubclass has been further characterized and delineated by specificradioimmunoassays and radioreceptor assays--e.g., antibodies raisedagainst purified CSF-1 are able to suppress specifically CSF-1 activity,without affecting the biological activities of the other subclasses, andmacrophage cell line J774 contains receptors which bind CSF-1specifically. A description of these assays was published by Das, S. K.,et al, Blood (1981) 58:630.

A significant difficulty in putting CSF proteins in general, and CSF-1in particular, to any useful function has been their unavailability indistinct and characterizable form in sufficient amounts to make theiremployment in therapeutic use practical or even possible. The presentinvention remedies these deficiencies by providing purified human andmurine CSF-1 in useful amounts through recombinant techniques.

(A CSF protein of a different subclass, murine and human GM-CSF, hasbeen purified and the cDNAs have been cloned. This protein was shown tobe distinct from other CSFs, e.g., CSF-1, by Gough, et al, Nature (1984)309:763-767. This GM-CSF protein is further described in W087/02060,published Apr. 9, 1987 as being useful to treat cancer patients toregenerate leukocytes after traditional cancer treatment, and to reducethe likelihood of viral, bacterial, fungal and parasitic infection, suchas acquired immune deficiency syndrome (AIDS). Murine IL-3 has beencloned by Fung, M. C., et al, Nature (1984) 307:233. See also Yokota,T., et al, Proc Natl Acad Sci (U.S.A.) (1984) 81:1070-1074; Wong, G. G.,et al, Science (1985) 228:810-815; Lee, F., et al, Proc Natl Acad Sci(U.S.A.) (1985) 82:4360-4364; and Cantrell, M. A., et al, Proc Natl AcadSci (U.S.A.) (1985) 82:6250-6254.)

Treatment of patients suffering from AIDS with CSF-1, alone or togetherwith erythropoietin and/or an antiviral agent and/or IL-2 is reported inW087/03204, published June 4, 1987. U.S. Pat. No. 4,482,485, issued Nov.13, 1984 states that CSF can be used for a supporting role in thetreatment of cancer. In addition, EP 118,915, published Sept. 19, 1984reports production of CSF for preventing and treating granulocytopeniaand macrophagocytopenia in patients receiving cancer therapy, forpreventing infections, and for treating patients with implanted bonemarrow. In addition, CSF-1 is reported to stimulate nonspecifictumoricidal activity (Ralph et al, Immunobiol (1986) 172:194-204). Ralphet al, Cell Immunol (1983) 76:10-21 reported that CSF has no immediatedirect role in activation of macrophages for tumoricidal andmicrobiocidal activities against fibrosarcoma 1023, lymphoma 18-8, andL. tropica amastigotes. Ralph et al, Cell Immunol (1987) 105:270-279reports the added tumoricidal effect of a combination of CSF-1 andlymphokine on murine sarcoma TU5 targets. Copending U.S. applicationSer. No. 948,159, filed Dec. 31, 1986, now abandoned discloses use ofCSF-1 and G-CSF.

In addition, Warren et al, J Immunol (1986) 137:2281-2285 discloses thatCSF-1 stimulates monocyte production of interferon, TNF and colonystimulating activity. Lee et al, J Immunol (1987) 138:3019-3022discloses CSF-1-induced resistance to viral infection in murinemacrophages.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to methods of enhancing productionof interferon, tumor necrosis factor and myeloid CSF from monocytescomprising treating said monocytes with an effective amount ofrecombinant CSF-1. In another aspect, the invention relates to methodsof enhancing the killing of target cells by macrophage, of enhancing theproduction of white blood cells from stem cells or enhancing the immunesystem of a subject, of inducing resistance to viral infections inmacrophages, of promoting wound healing, and of treating tumor cells byusing an effective amount of CSF-1. In addition, the invention relatesto pharmaceutical and therapeutic compositions comprising CSF-1 or amixture thereof with an excipient or a cytokine or lymphokine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1c show the partial amino acid sequences of human urinary andmurine L-929 cell CSF-1 as determined from purified native proteins.

FIG. 2 shows the sequence of certain oligomer probes for murine CSF-1.

FIG. 3 shows the sequence of oligomer probes used to obtain humangenomic CSF-1.

FIGS. 4a-4c show the sequenced portion of a 3.9 kb HindIII fragmentencoding human CSF-1 sequences and the deduced amino acid sequences forthe exon regions.

FIGS. 5-1 and 5-2 shows the DNA and deduced amino acid sequences for acDNA clone encoding CSF-1.

FIG. 6 shows a comparison of the activities of CSF-1 and other colonystimulating factors in enhancing the ability of macrophage to kill tumorcells.

FIGS. 7a-7b shows the results of sucrose gradient fractionation ofMIAPaCa mRNA.

MODES FOR CARRYING OUT THE INVENTION

A. Definitions

"Colony stimulating factor-1 (CSF-1)" refers to a protein which exhibitsthe spectrum of activity understood in the art for CSF-1--i.e., whenapplied to the standard in vitro colony stimulating assay of Metcalf,D., J Cell Physiol (1970) 76:89, it results in the formation ofprimarily macrophage colonies. Native CSF-1 is a glycosylated dimer;dimerization may be necessary for activity. Contemplated within thescope of the invention and within the definition of CSF-1 are both thedimer and monomeric forms. The monomeric form may be converted to thedimer by in vitro provision of intracellular conditions, and the monomeris per se useful as an antigen to produce anti-CSF-1 antibodies.

There appears to be some species specificity: Human CSF-1 is operativeboth on human and on murine bone marrow cells; murine CSF-1 does notshow activity with human cells. Therefore, "human" CSF-1 should bepositive in the specific murine radioreceptor assay of Das, S. K., etal, Blood (1981) 58:630, although there is not necessarily a completecorrelation. The biological activity of the protein will generally alsobe inhibited by neutralizing antiserum to human urinary CSF-1 (Das, S.K., et al, supra). However, in certain special circumstances (such as,for example, where a particular antibody preparation recognizes a CSF-1epitope not essential for biological function, and which epitope is notpresent in the particular CSF-1 mutein being tested) this criterion maynot be met.

Certain other properties of CSF-1 have been recognized more recently,including the ability of this protein to stimulate the secretion ofseries E prostaglandins, interleukin-1, and interferon from maturemacrophages (Moore, R., et al, Science (1984) 223:178). The mechanismfor these latter activities is not at present understood, and forpurposes of definition herein, the criterion for fulfillment of thedefinition resides in the ability to stimulate the formation ofmonocyte/macrophage colonies using bone marrow cells from theappropriate species as starting materials, under most circumstances (seeabove) the inhibition of this activity by neutralizing antiserum againstpurified human urinary CSF-1, and, where appropriate for species type, apositive response to the radioreceptor assay. (It is known that theproliferative effect of CSF-1 is restricted to cells of mononuclearphagocytic lineage (Stanley, E. R., The Lymphokines (1981), Stewart, W.E., II, et al, ed, Humana Press, Clifton, N.J.), pp. 102-132) and thatreceptors for CSF-1 are restricted to these cell lines (Byrne, P. V., etal, Cell Biol (1981) 91:848) and placental trophoblasts).

As is the case for all proteins, the precise chemical structure dependson a number of factors. As ionizable amino and carboxyl groups arepresent in the molecule, a particular protein may be obtained as anacidic or basic salt, or in neutral form. All such preparations whichretain their activity when placed in suitable environmental conditionsare included in the definition. Further, the primary amino acid sequencemay be augmented by derivatization using sugar moieties (glycosylation)or by other supplementary molecules such as lipids, phosphate, acetylgroups and the like, more commonly by conjugation with saccharides. Theprimary amino acid structure may also aggregate to form complexes, mostfrequently dimers. Indeed, native human urinary CSF-1 is isolated as ahighly glycosylated dimer of 45⁺ kd. Certain aspects of suchaugmentation are accomplished through post-translational processingsystems of the producing host; other such modifications may beintroduced in vitro. In any event, such modifications are included inthe definition so long as the activity of the protein, as defined above,is not destroyed. It is expected, of course, that such modifications mayquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the protein in the various assays.

Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the protein may becleaved to obtain fragments which retain activity. Such alterationswhich do not destroy activity do not remove the protein sequence fromthe definition.

Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the sequence duringtranslation can be made without destroying the activity of the protein.Such substitutions or other alterations result in proteins having anamino acid sequence which falls within the definition of proteins"having an amino acid sequence substantially equivalent to that ofCSF-1". Indeed, human- and murine-derived CSF-1 proteins havenon-identical but similar primary amino acid sequences which display ahigh homology.

For convenience, the mature protein amino acid sequence of the monomerprotein of a dimeric protein shown in FIG. 5, deduced from the cDNAclone illustrated herein, is designated mCSF-1 (mature CSF-1). FIG. 5shows the presence of a 32 residue putative signal sequence, which ispresumably cleaved upon secretion from mammalian cells; mCSF-1 isrepresented by amino acids 1-224 shown in that figure. Specificallyincluded in the definition of human CSF-1 are muteins which monomers anddimers are mCSF-1 and related forms of mCSF-1, designated by theirdifferences from mCSF-1. CSF-1 derived from other species may fit thedefinition of "human" CSF-1 by virtue of its display of the requisitepattern of activity as set forth above with regard to human substrate.

Also for convenience, the amino acid sequence of mCSF-1 will be used asa reference and other sequences which are substantially equivalent tothis in terms of CSF-1 activity will be designated by referring to thesequence shown in FIG. 5. The substitution of a particular amino acidwill be noted by reference to the amino acid residue which it replaces.Thus, for example, ser₉₀ CSF-1 refers to the protein which has thesequence shown in FIG. 5 except that the amino acid at position 90 isserine rather than cysteine. Deletions are noted by a ∇ followed by thenumber of amino acids deleted from the N-terminal sequence, or by thenumber of amino acids remaining when residues are deleted from theC-terminal sequence, when the number is followed by a minus sign. Thus,∇₄ CSF-1 refers to CSF-1 of FIG. 5 wherein the first 4 amino acids fromthe N-terminus have been deleted; ∇₁₃₀₋ refers to CSF-1 wherein the last94 amino acids following amino acid 130 have been deleted. Illustratedbelow are, for example, asp₅₉ CSF-1, which contains an aspartic residueencoded by the gene (FIG. 4) at position 59 rather than the tyrosineresidue encoded by the cDNA, and ∇₁₅₈₋ CSF-1, which comprises only aminoacids 1-158 of mCSF-1.

"Operably linked" refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence "operablylinked" to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequences.

"Control sequences" refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences which are suitable for procaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood, sequences.Eucaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

"Effective amount" signifies an amount effective to perform the functionspecified, such as to kill tumors or reduce tumor burden or prevent orcure infectious diseases.

"Therapeutic treatment" indicates treating after the disease iscontracted, whereas "prophylactic" treatment indicates treating beforethe disease is contracted.

"Mammals" indicates any mammalian species, and includes rabbits, mice,dogs, cats, primates and humans, preferably humans.

"Expression system" refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. In order to effect transformation, the expression system maybe included on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein "cell", "cell line", and "cell culture" are usedinterchangeably and all such designations include progeny. Thus"transformants" or "transformed cells" includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny which have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

B. General Description

The CSF-1 proteins of the invention are capable both of stimulatingmonocyte-precursor/macrophage cell production from progenitor marrowcells, thus enhancing the effectiveness of the immune system, and ofstimulating such functions of these differentiated cells as thesecretion of lymphokines in the mature macrophages.

In one application, these proteins are useful as adjuncts tochemotherapy. It is well understood that chemotherapeutic treatmentresults in suppression of the immune system. Often, although successfulin destroying the tumor cells against which they are directed,chemotherapeutic treatments result in the death of the subject due tothis side effect of the chemotoxic agents on the cells of the immunesystem. Administration of CSF-1 to such patients, because of the abilityof CSF-1 to mediate and enhance the growth and differentiation of bonemarrow-derived precursors into macrophages, results in a restimulationof the immune system to prevent this side effect, and thus to preventthe propensity of the patient to succumb to secondary infection. Otherpatients who would be helped by such treatment include those beingtreated for leukemia through bone marrow transplants; they are often inan immunosuppressed state to prevent rejection. For these patients also,the immunosuppression could be reversed by administration of CSF-1.

In general, any subject suffering from immunosuppression whether due tochemotherapy, bone marrow transplantation, or other, accidental forms ofimmunosuppression such as disease (e.g., acquired immune deficiencysyndrome) and wounds would benefit from the availability of CSF-1 forpharmacological use. In addition, subjects could be supplied enhancedamounts of previously differentiated macrophages to supplement those ofthe indigenous system, which macrophages are produced by in vitroculture of bone marrow or other suitable preparations treated withCSF-1. These preparations include those of the patient's own bloodmonocytes, which can be so cultured and returned for local or systemictherapy.

The ability of CSF-1 to stimulate production of lymphokines bymacrophages and to enhance their ability to kill target cells also makesCSF-1 directly useful in treatment of neoplasms and infections.

CSF-1 stimulates the production of interferons by murine-derivedmacrophage (Fleit, H. B., et al, J Cell Physiol (1981) 108:347), andhuman, partially purified, CSF-1 from MIAPaCa cells stimulates thepoly(I):poly(C)-induced production of interferon and TNF from humanmonocytes as illustrated below. In addition, CSF-1 stimulates theproduction of myeloid CSF by human blood monocytes.

Moreover, CSF-1 can be employed in conjunction with another lymphokineor cytokine such as, e.g., α-IFN, β-IFN, γ-IFN, IL-1, IL-2, IL-3, IL-4,G-CSF, or TNF to treat tumors.

Also illustrated below is a demonstration of the ability of murine CSF-1(from L-cell-conditioned medium) to stimulate normal C3H/HeN mouseperitoneal macrophages to kill murine sarcoma TU5 targets. This activityis most effective when the CSF-1 is used as pretreatment and during theeffector phase. The ability of CSF-1 to do so is much greater than thatexhibited by other colony stimulating factors, as shown in FIG. 6hereinbelow.

In addition, the ability of murine cells to attack infectious organisms,including viruses such as cytomegalovirus (CMV), fungi, and bacterialagents causing Gram-negative septis, is enhanced by CSF-1. (Murine CSF-1is inconsistently reported to stimulate murine macrophage to becytostatic to P815 tumor cells (Wing, E. J., et al, J Clin Invest (1982)69:270) or not to kill other leukemia targets (Ralph, P, et al, CellImmunol (1983) 76:10). Nogawa, R. T., et al, Cell Immunol (1980) 53:116,report that CSF-1 may stimulate macrophage to ingest and kill yeast.)

Thus, in addition to overcoming immunosuppression per se, CSF-1 can beused to destroy the invading organisms or malignant cells indirectly bystimulation of macrophage secretions and activity.

CSF-1 may also be used to cure leukopenia, a disease involving adeficiency in the total number of while blood cells. Neutropeniareflects a deficiency affecting principally the polymorphonuclearleukocytes (neutrophil, granulocytes) and may be due to variousinfections, certain drugs (e.g., cytotoxic drugs) or ionizingradiations. Thus, in vivo administration of CSF-1 can be used to inducestem cells to increase circulation of white blood cell count andneutrophil granulocyte count.

Finally, the CSF-1 may be used to promote wound healing when appliedeither locally or systemically and may recruit macrophages, as well asinduce them to produce tumor necrosis factor (TNF), platelet-derivedgrowth factor (PDGF) and other connective tissue growth factors.

The CSF-1 of the invention may be formulated in conventional waysstandard in the art for the administration of protein substances.Administration by injection is preferred; formulations include solutionsor suspensions, emulsions, or solid composition for reconstitution intoinjectables. Suitable excipients include, for example, Ringer'ssolution, Hank's solution, water, saline, glycerol, dextrose solutions,and the like. In addition, the CSF-1 of the invention may bepreincubated with preparations of cells in order to stimulateappropriate responses, and either the entire preparation or thesupernatant therefrom introduced into the subject. As shown hereinbelow,the materials produced in response to CSF-1 stimulation by various typesof blood cells are effective against desired targets, and the propertiesof these blood cells themselves to attack invading viruses or neoplasmsmay be enhanced. The subject's own cells may be withdrawn and used inthis way, or, for example, monocytes or lymphocytes from anothercompatible individual employed in the incubation.

Although the existence of a pattern of activity designated CSF-1 hasbeen known for some time, the protein responsible has never beenobtained in both sufficient purity and in sufficient amounts to permitsequence determination, nor in sufficient purity and quantity to providea useful therapeutic function. Because neither completely pure practicalamounts of the protein nor its encoding DNA have been available, it hasnot been possible to optimize modifications to structure by providingsuch alternatives as those set forth in A above, nor has it beenpossible to utilize this protein in a therapeutic context.

The present invention remedies these defects. Through a variety ofadditional purification procedures, sufficient pure CSF-1 has beenobtained from human urine to provide some amino acid sequence, thuspermitting the construction of DNA oligomeric probes. The probes areuseful in obtaining the coding sequence for the entire protein. Oneapproach, illustrated below, employs probes designed with respect to thehuman N-terminal sequence to probe the human genomic library to obtainthe appropriate coding sequence portion. The human genomic clonedsequence can be expressed directly using its own control sequences, orin constructions appropriate to mammalian systems capable of processingintrons. The genomic sequences are also used as probes for a human cDNAlibrary obtained from a cell line which produces CSF-1 to obtain cDNAencoding this protein. The cDNA, when suitably prepared, can beexpressed directly in COS or CV-1 cells and can be constructed intovectors suitable for expression in a wide range of hosts.

Thus these tools can provide the complete coding sequence for humanCSF-1 from which expression vectors applicable to a variety of hostsystems can be constructed and the coding sequence expressed. Thevariety of hosts available along with expression vectors suitable forsuch hosts permits a choice among post-translational processing systems,and of environmental factors providing conformational regulation of theprotein thus produced.

C. Suitable Hosts, Control Systems and Methods

In general terms, the production of a recombinant form of CSF-1typically involves the following:

First a DNA is obtained that encodes the mature (used here to includeall muteins) protein, the preprotein, or a fusion of the CSF-1 proteinto an additional sequence which does not destroy its activity or toadditional sequences cleavable under controlled conditions (such astreatment with peptidase) to give an active protein. If the sequence isuninterrupted by introns it is suitable for expression in any host. Ifthere are introns, expression is obtainable in mammalian or othereucaryotic systems capable of processing them. This sequence should bein excisable and recoverable form.

The excised or recovered coding sequence is then preferably placed inoperable linkage with suitable control sequences in a replicableexpression vector. The vector is used to transform a suitable host andthe transformed host cultured under favorable conditions to effect theproduction of the recombinant CSF-1. Optionally the CSF-1 is isolatedfrom the medium or from the cells; recovery and purification of theprotein may not be necessary in some instances, where some impuritiesmay be tolerated. For example, for in vitro cultivation of cells fromwhich a lymphokine factor will be isolated for administration to asubject, complete purity is not required. However, direct use in therapyby administration to a subject would, of course, require purification ofthe CSF-1 produced.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences can be obtained by preparingsuitable cDNA from cellular messenger and manipulating the cDNA toobtain the complete sequence. Alternatively, genomic fragments may beobtained and used directly in appropriate hosts. The constructions forexpression vectors operable in a variety of hosts are made usingappropriate replicons and control sequences, as set forth below.Suitable restriction sites can, if not normally available, be added tothe ends of the coding sequence so as to provide an excisable gene toinsert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, insect, or mammalian cells are presentlyuseful as hosts. Since native CSF-1 is secreted as a glycosylated dimer,host systems which are capable of proper post-translational processingare preferred. Accordingly, although procaryotic hosts are in generalthe most efficient and convenient for the production of recombinantproteins, eucaryotic cells, and, in particular, mammalian cells orinsect cells are preferred for their processing capacity. RecombinantCSF-1 produced by bacteria would require in vitro dimerization. Inaddition, there is more assurance that the native signal sequence willbe recognized by mammalian cell or insect cell hosts making secretionpossible, and therefore purification easier.

In the particular case of human CSF-1, evidence now accumulatingindicates that considerable deletion at the C-terminus of the proteinmay occur under both recombinant and native conditions, and that theactivity of the protein is still retained. It appears that the nativeproteins isolated may be in some sort of C-terminal truncated form ormixtures thereof, and may exhibit variable C-terminal processing. Theactivity of these "truncated" forms is clearly established by theirdeliberate production. The mutein produced from DNA encoding SCSF/C∇150,for example, is fully active in assays for CSF-1, as is that producedfrom cDNA encoding LCSF/C∇190. These are described in copending U.S.Ser. No. 039,654, filed Apr. 16, 1987 now abandoned, and incorporatedherein by reference. The products of recombinant expression of both longand short forms of the genes seem to exhibit molecular weights lowerthan would be expected from the full length sequence. It is believedthat "natural" processing may occur at a variety of proteolytic sites,including, for example in the long form, at the lys residue at 238, thearg residue at 249, and the arg at 411. Since it is clear that these Cterminal shortened forms are active, the constructs used may alsoinclude the corresponding shortened forms of the coding sequence.

C.1. Control Sequences And Corresponding Hosts

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example, Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al, Gene (1977) 2:95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences, which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al, Nature (1977) 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al Nucleic Acids Res (1980) 8:4057) and the lambdaderived P_(L) promoter (Shimatake, et al, Nature (1981) 292:128), andN-gene ribosome binding site, which has been made useful as a portablecontrol cassette, (as set forth in copending Application Ser. No.685,312, filed Dec. 24, 1984), which comprises a first DNA sequence thatis the pL promoter operably linked to a second DNA sequencecorresponding to the N_(RBS) upstream of a third DNA sequence having atleast one restriction site that permits cleavage within 6 bp 3' of theN_(RBS) sequence. Also useful is the phosphatase A (phoA) systemdescribed by Chang et al. in European Patent Publication No. 196,864,published Oct. 8, 1986, incorporated herein by reference. However, anyavailable promoter system compatible with procaryotes can be used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used, although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated (Broach, J. R., Meth Enz (1983) 101:307), other plasmidvectors suitable for yeast expression are known (see, for example,Stinchcomb, et al, Nature (1979) 282:39, Tschempe, et al, Gene (1980)10:157 and Clarke, L., et al, Meth Enz (1983) 101:300). Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess, et al, J Adv Enzyme Reg (1968) 7:149; Holland,et al, Biochemistry (1978) 17:4900).

Additional promoters known in the art include the promoter for3-phosphoglycerate kinase (Hitzeman, et al, J Biol Chem (1980)255:2073), and those for other glycolytic enzymes, such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters, which havethe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization (Holland, ibid).

It is also believed that terminator sequences are desirable at the 3'end of the coding sequences. Such terminators are found in the 3'untranslated region following the coding sequences in yeast-derivedgenes. Many of the vectors illustrated contain control sequences derivedfrom the enolase gene containing plasmid peno46 (Holland, M. J., et al,J Biol Chem (1981) 256:1385) or the LEU2 gene obtained from YEp13(Broach, J., et al, Gene (1978) 8:121); however, any vector containing ayeast compatible promoter, origin of replication and other controlsequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, Academic Press, Cruz and Patterson,editors (1973). Useful host cell lines include murine myelomas N51, VEROand HeLa cells, and Chinese hamster ovary (CHO) cells. Expressionvectors for such cells ordinarily include promoters and controlsequences compatible with mammalian cells such as, for example, thecommonly used early and late promoters from Simian Virus 40 (SV 40)(Fiers, et al, Nature (1978) 273:113), or other viral promoters such asthose derived from polyoma, Adenovirus 2, bovine papiloma virus, oravian sarcoma viruses, or immunoglobulin promoters and heat shockpromoters. A system for expressing DNA in mammalian systems using theBPV as a vector is disclosed in U.S. Pat. No. 4,419,446. A modificationof this system is described in U.S. Pat. No. 4,601,978. General aspectsof mammalian cell host system transformations have been described byAxel; U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. It now appears alsothat "enhancer" regions are important in optimizing expression; theseare, generally, sequences found upstream of the promoter region. Originsof replication may be obtained, if needed, from viral sources. However,integration into the chromosome is a common mechanism for DNAreplication in eucaryotes.

Plant cells are also now available as hosts, and control sequencescompatible with plant cells such as the nopaline synthase promoter andpolyadenylation signal sequences (Depicker, A., et al, J Mol Appl Gen(1982) 1:561) are available.

Recently, in addition, expression systems employing insect cellsutilizing the control systems provided by baculovirus vectors have beendescribed (Miller, D. W., et al, in Genetic Engineering (1986) Setlow,J. K. et al. eds., Plenum Publishing, Vol. 8, pp. 277-279 and copendingU.S. Ser. No. 077,188, filed July 24, 1987 now abandoned incorporatedherein by reference). These systems are also successful in producingCSF-1.

C.2. Transformations

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., Proc Natl Acad Sci(U.S.A.) (1972) 69:2110, is used for procaryotes or other cells whichcontain substantial cell wall barriers. Infection with Agrobacteriumtumefaciens (Shaw, C. H., et al, Gene (1983) 23:315) is used for certainplant cells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, Virology (1978)52:546 is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P., et al, J Bact (1977)130:946 and Hsiao, C. L., et al, Proc Natl Acad Sci (U.S.A.) (1979)76:3829.

C.3. Probing mRNA by Northern Blot; Probe of cDNA or Genomic Libraries

RNA is fractionated for Northern blot by agarose slab gelelectrophoresis under fully denaturing conditions using formaldehyde(Maniatis, T., et al, Molecular Cloning (1982) Cold Spring Harbor Press,pp 202-203) or 10 mM methyl mercury (CH₃ HgOH) (Bailey, J. M., et al,Anal Biochem (1976) 70:75-85; and Sehgal, P. B., et al, Nature (1980)288:95-97) as the denaturant. For methyl mercury gels, 1.5% gels areprepared by melting agarose in running buffer (100 mM boric acid, 6 mMsodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60°C. and adding 1/100 volume of 1M CH₃ HgOH. The RNA is dissolved in 0.5×running buffer and denatured by incubation in 10 mM methyl mercury for10 min at room temperature. Glycerol (20%) and bromophenol blue (0.05%)are added for loading the samples. Samples are electrophoresed for500-600 volt-hr with recirculation of the buffer. After electrophoresis,the gel is washed for 40 min in 10 mM 2-mercaptoethanol to detoxify themethyl mercury, and Northern blots are prepared by transferring the RNAfrom the gel to a membrane filter.

cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Bacterial colonies, or the plaques for phage,are lifted onto duplicate nitrocellulose filter papers (S & S typeBA-85). The plaques or colonies are lysed and DNA is fixed to the filterby sequential treatment for 5 min with 500 mM NaOH, 1.5M NaCl. Thefilters are washed twice for 5 min each time with 5×standard salinecitrate (SSC) and are air dried and baked at 80° C. for 2 hr.

The gels for Northern blot or the duplicate filters for cDNA or genomicscreening are prehybridized at 25°-42° C. for 6-8 hr with 10 ml perfilter of DNA hybridization buffer without probe (0-50% formamide,5-6×SSC, pH 7.0, 5×Denhardt's solution (polyvinylpyrrolidine, plusFicoll and bovine serum albumin; 1×=0.02% of each), 20-50 mM sodiumphosphate buffer at pH 7.0, 0.2% SDS, 20 μg/ml poly U (when probingcDNA), and 50 μg/ml denatured salmon sperm DNA). The samples are thenhybridized by incubation at the appropriate temperature for about 24-36hours using the hybridization buffer containing kinased probe (foroligomers). Longer cDNA or genomic fragment probes were labeled by nicktranslation or by primer extension.

The conditions of both prehybridization and hybridization depend on thestringency desired, and vary, for example, with probe length. Typicalconditions for relatively long (e.g., more than 30-50 nucleotide) probesemploy a temperature of 42°-55° C. and hybridization buffer containingabout 20%-50% formamide. For the lower stringencies needed foroligomeric probes of about 15 nucleotides, lower temperatures of about25°-42° C., and lower formamide concentrations (0%-20%) are employed.For longer probes, the filters may be washed, for example, four timesfor 30 minutes, each time at 40°-55° C. with 2×SSC, 0.2% SDS and 50 mMsodium phosphate buffer at pH 7, then washed twice with 0.2×SSC and 0.2%SDS, and air dried, and are autoradiographed at -70° C. for 2 to 3 days.Washing conditions are somewhat less harsh for shorter probes.

C.4. Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μlof buffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology (1980)65:499-560.

Restriction-cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5'sticky ends but chews back protruding 3' single strands, even though thefour dNTPs are present. If desired, selective repair can be performed bysupplying only one of the, or selected, dNTPs within the limitationsdictated by the nature of the sticky ends. After treatment with Klenow,the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with S1 nucleaseresults in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared by the triester method ofMatteucci, et al (J Am Chem Soc (1981) 103:3185-3191) or using automatedsynthesis methods. Kinasing of single strands prior to annealing or forlabeling is achieved using an excess, e.g., approximately 10 units ofpolynucleotide kinase to 1 nmole substrate in the presence of 50 mMTris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol, 1-2 mM ATP. If kinasingis for labeling of probe, the ATP will contain high specific activityγ-³² P.

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for "sticky end" ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

In vector construction employing "vector fragments", the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per μg of vector at60° for about one hour. In order to recover the nucleic acid fragments,the preparation is extracted with phenol/chloroform and ethanolprecipitated. Alternatively, religation can be prevented in vectorswhich have been double digested by additional restriction enzymedigestion of the unwanted fragments.

C.5. Modification of DNA Sequences

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site-specific primer directed mutagenesis isused. This technique is now standard in the art, and is conducted usinga primer synthetic oligonucleotide complementary to a single strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells which harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The plaques are transferred to nitrocellulose filters and the "lifts"hybridized with kinased synthetic primer at a temperature which permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques whichhybridize with the probe are then picked and cultured, and the DNA isrecovered. Details of site specific mutation procedures are describedbelow in specific examples.

C.6. Verification of Construction

In the constructions set forth below, correct ligations for plasmidconstruction are confirmed by first transforming E. coli strain MM294,or other suitable host, with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers, depending on the mode ofplasmid construction, as is understood in the art. Plasmids from thetransformants are then prepared according to the method of Clewell, D.B., et al, Proc Natl Acad Sci (U.S.A.) (1969) 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., J. Bacteriol(1972) 110:667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al, Proc Natl Acad Sci(U.S.A.) (1977) 74:5463 as further described by Messing, et al, NucleicAcids Res (1981) 9:309, or by the method of Maxam, et al, Methods inEnzymology (1980) 65:499.

C.7. Hosts Exemplified

Host strains used in cloning and expression herein are as follows:

For cloning and sequencing, and for expression of construction undercontrol of most bacterial promoters, E. coli strain MM294 obtained fromE. coli Genetic Stock Center GCSC #6135, was used as the host. Forexpression under control of the P_(L) N_(RBS) promoter, E. coli strainK12 MC1000 lambda lysogen, N₇ N₅₃ cI857 SusP₈₀, ATCC 39531 may be used.Also, E. coli DG116, which was deposited with ATCC (ATCC 53606) on Apr.7, 1987, may be used.

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98, are employed. The DG98strain has been deposited with ATCC on July 13, 1984 and has accessionnumber 39768.

Mammalian expression has been accomplished in COS-7 and CV-1 cells, andalso can be accomplished in COS-A2, hamster, and murine cells. Insectcell-based expression can be in Spodoptera frugipeida.

D. Preferred Embodiments

The recombinant CSF-1 of the invention can be considered a set ofmuteins which have similar but not necessarily identical primary aminoacid sequences, all of which exhibit, or are specifically cleavable to amutein which exhibits, the activity pattern characteristic ofCSF-1--i.e. they are capable of stimulating bone marrow cells todifferentiate into monocytes, preponderantly, and, within thelimitations set forth in the Definitions section above, areimmunoreactive with antibodies raised against native CSF-1 and with thereceptors associated with CSF-1 activity. Certain embodiments of thesemuteins are, however, preferred.

The primary sequence shown in FIG. 5 for mCSF-1 has the requiredactivity, and it is, of course, among the preferred embodiments. Alsopreferred are muteins wherein certain portions of the sequences havebeen altered by either deletion of, or conservative substitution of, oneor more amino acids in mCSF-1. By a "conservative" amino acidsubstitution is meant one which does not change the activitycharacteristics of the protein, and in general is characterized bychemical similarity of the side chains of the two residues interchanged.For example, acidic residues are conservatively replaced by other acidicresidues, basic by basic, hydrophobic by hydrophobic, bulky by bulky,and so forth. The degree of similarity required depends, of course, onthe criticality of the amino acid for which substitution is made, andits nature. Thus, in general, preferred substitutions for cysteineresidues are serine and alanine; for aspartic acid residues, glutamicacid; for lysine or arginine residues, histidine; for leucine residues,isoleucine, or valine; for tryptophan residues, phenylalanine ortyrosine; and so forth.

Regions of the CSF-1 protein which are most tolerant of alterationinclude those regions of known low homology between human and mousespecies (residues 15-20 and 75-84); regions which confer susceptibilityto proteolytic cleavage (residues 51 and 52 and residues 191-193);cysteine residues not participating in disulfide linkages, or otherresidues which are not absolutely essential for activity (residues159-224). It also appears residues 151-224 are not essential.

Therefore, particularly preferred are those CSF-1 muteins characterizedby the deletion or conservative substitution of one or more amino acidsand/or one or more sequences of amino acids between positions 159 and224 inclusive of mCSF-1 or positions 151-224 inclusive. In particular,∇₁₅₈₋ CSF-1 has CSF activity comparable to that of the native protein,even if a limited number of additional amino acid residues not relatedto CSF-1 are included at the truncated C-terminus; ∇₁₅₀₋ CSF-1 hassimilar activity. Indeed, the native protein is reported to have amolecular weight of 14-15 kd (as opposed to the 26 kd predicted from thecDNA sequence) and the hydrophobicity deduced from the recombinant(predicted) amino acid sequence corresponds to a transmembrane regionnormally susceptible to cleavage. It may, therefore, be that thetruncated version corresponds in a rough way to the CSF-1 as isolated.

Also preferred are muteins characterized by the deletion or conservativesubstitution of one or more of the amino acids at positions 51 and 52and/or positions 191, 192 and 193 of mCSF-1. Especially preferred isgln₅₂ CSF-1; a corresponding proline substitution is not conservative,and does not yield an active CSF. Since they represent regions ofapparently low homology, another preferred set of embodiments is thatcharacterized by the deletion or conservative substitution of one ormore of the amino acids at positions 15-20 and/or positions 75-84 ofmCSF-1. Also preferred are those muteins characterized by the deletionor conservative substitution of the cysteine residue at any position notessential for disulfide bond formation. Also preferred are those muteinscharacterized by the deletion or substitution of the tyrosine residue atposition 59 of mCSF-1; particularly, substitution by an aspartic acidresidue.

E. Cloning and Expression of Human CSF-1

The following illustrates the methods used in obtaining the codingsequence for human CSF-1, for disposing this sequence in expressionvectors, and for obtaining expression of the desired protein.

E.1. Purification of Native Human CSF-1 and Probe Design

Human urinary CSF-1 was partially purified by standard methods asdescribed by Das, S. K., et al, Blood (1981) 58:630, followed by anaffinity purification step using a rat monoclonal antibody to murineCSF-1, designated YYG106, attached to a Sepharose 4B column (Stanley, E.R., Methods Enzymol (1985) 116:564). The final step in purification wasreverse phase HPLC in a 0.1% TFA/30% acetonitrile--0.1% TFA/60%acetonitrile buffer system.

For MIAPaCa CSF-1, which was produced serum-free by induction withphorbol myristic acetate, the cell supernatant was subjected to calciumphosphate gel chromatography (according to Das (supra)), followed byaffinity chromatography using lentil lectin (in place of the ConAaffinity step of Das), and then to the immunoaffinity step employing theYYG106 monoclonal antibody conjugated to Sepharose 4B and to the reversephase HPLC, both as above described.

The urinary and MIAPaCa proteins, having been purified to homogeneity,were subjected to amino acid sequencing using Edman degradation on anautomated sequencer. Sufficient N-terminal sequence of human CSF wasdetermined to permit construction of probes shown in FIG. 3.

E.2. Preparation of the Human Genomic Sequence

A human genomic sequence encoding CSF-1 was obtained from the Maniatishuman genomic library in λ phage Charon 4 using probes designed toencode the N-terminal sequence of human protein. The library wasconstructed using partial HaeIII/AluI digestion of the human genome,ligation to EcoRI linkers, and insertion of the fragments into EcoRIdigested Charon 4 phage. A Charon 4A phage containing the CSF-1 sequenceas judged by hybridization to probe as described below, and designatedpHCSF-1, was deposited with the American Type Culture Collection (ATCC)on Apr. 2, 1985 and has accession no. 40177. Upon later study of thisphage, it was found that rearrangements and/or deletions had occurredand the correct sequences were not maintained. Therefore, an alternativecolony obtained from the genomic library in identical fashion, andpropagated to confirm stability through replication, was designatedpHCSF-1a and was deposited with ATCC on May 21, 1985, and givenaccession number 40185. pHCSF-1a contained an 18 kb insert and wascapable of generating restriction enzyme digests which also hybridizedto probe, and was used for sequence determination and additional probeconstruction as outlined below.

If the CSF-1 encoding sequence is present in its entirety its presencecan be demonstrated by expression in COS-7 cells, as described byGluzman, Y., Cell (1981) 23:175. The test fragment is cloned into aplasmid derived from pBR322 which has been modified to contain the SV40origin of replication (pGRI Ringold, G., J Mol Appl Genet (1982)1:165-175). The resulting high copy number vectors are transformed intoCOS-7 cells and expression of the CSF-1 gene is assayed after 24, 48,and 72 hours by the radioreceptor assay method described by Das (supra).Expression is under control of the native CSF-1 control sequences. TheHindIII digests of the approximately 18 kb insert of pHCSF-1a tested inthis manner failed to express, thus indicating that HindIII digests intothe gene. This was confirmed by subsequent mapping.

However, for initial sequencing, a 3.9 kb HindIII fragment was obtainedfrom the pHCSF-1a phage and cloned into M13 cloning vectors.

The HindIII fragment has been partially sequenced, and the results areshown in FIG. 4, along with a deduced peptide sequence. It contains thecorrect codons for the portion of the human CSF-1 protein for which theamino acid sequence had been determined, as set forth in FIG. 1. Thepresence of an intron of approximately 1400 bp was deduced from theavailable amino acid sequence. In addition, based on the genomicsequence encoding amino acids 24-34 (see overlined portion of FIGS. 4and 5), a 32-mer probe for the cDNA library was constructed and employedas described below.

In more detail, to obtain the genomic clone, pHCSF-1a, the Maniatislibrary was probed using two mixtures of oligomers shown in FIG. 3. EK14and EK15 were selected, although the other oligomers shown are useful aswell. A "full length" probe for the N-terminal sequence, EK14, was usedas a mixture of sixteen 35-mers. A shorter oligomer, EK15, was employedas a mixture of sixty-four 18-mers. Phage hybridizing to both kinasedprobes were picked and cultured by infection of E. coli DG98 or othercompetent strain.

Specific conditions for probing with EK14 and EK15 are as follows: forEK14, the buffer contained 15% formamide, 6×SSC, pH 7.0, 5× Denhardt's,20 mM sodium phosphate, 0.2% SDS and 50 μg/ml denatured salmon spermDNA. Prehybridization and hybridization were conducted at 42° C. and thefilters were washed in 2×SSC at 52° C. For EK15, similar conditions wereused for hybridization and prehybridization except for the formamideconcentration, which was 0%; washing was at a slightly lowertemperature, 42° C.

The approximately 18 kb DNA insert isolated from the positivelyhybridizing phage pHSCF-1a was treated with HindIII and the fragmentswere subjected to electrophoresis on agarose gel according to the methodof Southern. The gels were replicated onto nitrocellulose filters andthe filters were probed again with EK14 and EK15. Both probes hybridizedto a 3.9 kb fragment.

The positive fragment was excised from the gel, eluted, and subclonedinto HindIII-treated M13mp19 for dideoxy sequencing. A partial sequenceis shown in FIG. 4. The underlining corresponds precisely to thepreviously determined N-terminal sequence of human CSF-1; the residueswith dot subscripts are homologous to the murine sequence.

In FIG. 4, the 1.4 kb intron region between the codons for amino acids22 and 23, as deduced from the human sequence determined from thepurified protein, is shown untranslated. The sequence upstream of theN-terminal residues contains the putative leader; the translation of theportion of this leader immediately adjacent to the mature protein, whichwas tentatively verified by the preliminary results of sequencing of thecDNA clone (see below) is shown. The upstream portions are, however, notshown translated; these portions are confirmed by comparison to the cDNAto comprise an intron.

Further sequencing to obtain about 13 kb of the entire 18 kb gene showsthat the gene contains 9 exons separated by 8 introns. The regions ofthe mature protein cDNA correspond exactly to the genomic exon codonsexcept for codon 59, as further described below.

An additional M13 subclone was obtained by digestion of the HindIII 3.9kb fragment with PstI to generate a 1 kb PstI/PstI fragment whichincludes the known N-terminal sequence and about 1 kb of additionalupstream sequence.

E. 3. cDNA Encoding Human CSF-1

pcCSF-17

The human derived pancreatic carcinoma cell line MIAPaCa-2 was used as asource of mRNA to validate probes and for the formation of a cDNAlibrary containing an intronless form of the human CSF-1 codingsequence. The MIAPaCa cell line produces CSF-1 at a level approximately10 fold below that of the murine L-929 cells.

Negative control mRNA was prepared from MIAPaCa cells maintained inserum-free medium, i.e. under conditions wherein they do not produceCSF-1. Cells producing CSF-1 were obtained by reinducing CSF-1production after removal of the serum.

Cells were grown to confluence in roller bottles using Dulbecco'sModified Eagles' Medium (DMEM) containing 10% fetal calf serum, andproduce CSF-1 at 2000-6000 units/ml. The cell cultures were washed, andreincubated serum-free to suppress CSF-1 formation. For negativecontrols, no detectable CSF-1 was produced after a day or two. Reinducedcells were obtained by addition of phorbol myristic acetate (100 ng/ml)to obtain production after several days of 1000-2000 units/ml.

The mRNA was isolated by lysis of the cell in isotonic buffer with 0.5%NP-40 in the presence of ribonucleoside vanadyl complex (Berger, S. L.,et al, Biochemistry (1979) 18:5143) followed by phenol chloroformextraction, ethanol precipitation, and oligo dT chromatography, and anenriched mRNA preparation was obtained. In more detail, cells are washedtwice in PBS (phosphate buffered saline) and are resuspended in IHB (140mM NaCl, 10 mM Tris, 1.5 mM MgCl₂, pH 8) containing 10 mM vanadyladenosine complex (Berger, S. L., et al, supra).

A non-ionic detergent of the ethylene oxide polymer type (NP-40) isadded to 0.5% to lyse the cellular, but not nuclear membranes. Nucleiare removed by centrifugation at 1,000× g for 10 min. The post-nuclearsupernatant is added to two volumes of TE (10 mM Tris, 1 mMethylenediaminetetraacetic acid (EDTA), pH 7.5) saturated phenolchloroform (1:1) and adjusted to 0.5% sodium dodecyl sulfate (SDS) and10 mM EDTA. The supernatant is re-extracted 4 times and phase separatedby centrifugation at 2,000× g for 10 min. The RNA is precipitated byadjusting the sample to 0.25 M NaCl, adding 2 volumes of 100% ethanoland storing at -20° C. The RNA is pelleted at 5,000× g for 30 min, iswashed with 70% and 100% ethanol, and is then dried. Polyadenylated(poly A⁺) messenger RNA (mRNA) is obtained from the total cytoplasmicRNA by chromatography on oligo dT cellulose (Aviv, J., et al, Proc NatlAcad Sci (1972) 62:1408-1412). The RNA is dissolved in ETS (10 mM Tris,1 mM EDTA, 0.5% SDS, pH 7.5) at a concentration of 2 mg/ml. Thissolution is heated to 65° C. for 5 min, then quickly chilled to 4° C.After bringing the RNA solution to room temperature, it is adjusted to0.4M NaCl and is slowly passed through an oligo dT cellulose columnpreviously equilibrated with binding buffer (500 mM NaCl, 10 mM Tris, 1mM EDTA, pH 7.5 0.05% SDS). The flow-through is passed over the columntwice more. The column is then washed with 10 volumes of binding buffer.Poly A⁺ mRNA is eluted with aliquots of ETS, extracted once withTE-saturated phenol chloroform and is precipitated by the addition ofNaCl to 0.2M and 2 volumes of 100% ethanol. The RNA is reprecipitatedtwice, and is washed once in 70% and then in 100% ethanol prior todrying.

Total mRNA was subjected to 5-20% by weight sucrose gradientcentrifugation in 10 mM Tris HCl, pH 7.4, 1 mM EDTA, and 0.5% SDS usinga Beckman SW40 rotor at 20° C. and 27,000 rpm for 17 hr. The mRNAfractions were then recovered from the gradient by ethanolprecipitation, and injected into Xenopus oocytes in the standardtranslation assay. The oocyte products of the RNA fractions were assayedin the bone marrow assay (or to the bone marrow proliferation assays ofMoore, R. N., et al, J Immunol (1983) 131:2374, and of Prystowsky, M.B., et al, Am J Pathol (1984) 114:149) and the fractions themselves wereassayed by dot blot hybridization to a 32-mer probe corresponding to theDNA in the second exon of the genomic sequence (exon II probe). (Theoverlining in FIGS. 4 and 5 shows the exon II probe.) These results aresummarized in FIG. 7.

The broken line in FIG. 7A shows the response in the bone marrowproliferation assay of the supernatants from the Xenopus oocytes; FIGS.7B shows the dot-blot results. The most strongly hybridizing fraction,11, corresponds to 18S, while the most active fractions 8 and 9correspond to 14-16S. Fractions 8, 9 and 11 were used to form anenriched cDNA library as described below.

(The mRNA was also fractionated on a denaturing formaldehyde gel,transferred to nitrocellulose, and probed with exon II probe. Severaldistinct species ranging in size from 1.5 kb to 4.5 kb were found, evenunder stringent hybridization conditions. To eliminate the possibilityof multiple genes encoding CSF-1, digests of genomic DNA with variousrestriction enzymes were subjectd to Southern blot and probed usingpcCSF-17 DNA. The restriction pattern was consistent with the presenceof only one gene encoding CSF-1.)

The enriched mRNA pool was prepared by combining the mRNA from thegradient fractions having the highest bone marrow proliferativeactivity, although their ability to hybridize to probe is relatively low(14S-16S) with the fractions hybridizing most intensely to probe (18S).Higher molecular weight fractions which also hybridized to exon II probewere not included because corresponding mRNA from uninduced MIAPaCacells also hybridized to exonII probe.

cDNA libraries were prepared from total or enriched human mRNA in twoways. One method uses λgt10 phage vectors and is described by Huynh, T.V., et al, in DNA Cloning Techniques: A Practical Approach IRL Press,Oxford 1984, D. Glover, Ed.

A preferred method uses oligo dT priming of the poly A tails and AMVreverse transcriptase employing the method of Okayama, H., et al, MolCell Biol (1983) 3:280-289, incorporated herein by reference. Thismethod results in a higher proportion of full length clones than doespoly dG tailing and effectively uses as host vector portions of twovectors therein described, and readily obtainable from the authors,pcDV1 and pL1. The resulting vectors contain the insert between vectorfragments containing proximal BamHI and XhoI restriction sites; thevector contains the pBR322 origin of replication, and Amp resistancegene and SV40 control elements which result in the ability of the vectorto effect expression of the inserted sequences in COS-7 cells.

A 300,000 clone library obtained from the above enriched MIAPaCa mRNA bythe Okayama and Berg method was then probed under conditions of highstringency, using the exon II probe. Ten colonies hybridizing to theprobe were picked and colony purified. These clones were assayed for thepresence of CSF-1 encoding sequences by transient expression in COS-7cells. The cloning vector, which contains the SV40 promoter, was usedper se in the transformation of COS-7 cells.

Plasmid DNA was purified from the 10 positive clones using a CsClgradient, and the COS-7 cells are transfected using a modification(Wang, A. M., et al, Science (1985) 228:149) of the calcium phosphatecoprecipitation technique. After incubation for three days, CSF-1production was assayed by subjecting the culture supernatants to theradioreceptor assay performed substantially as disclosed by Das, S. K.,et al, Blood (1981) 58:630, and to a colony stimulation (bone marrowproliferation) assay performed substantially as disclosed by Prystowsky,M. B., et al, Am J Pathol (1984) 114: 149. Nine of the ten clones pickedfailed to show transient CSF-1 production in COS-7 cells. One clone,which did show expression, was cultured, the plasmid DNA isolated, andthe insert sequenced. The DNA sequence, along with the deduced aminoacid sequence, are shown in FIG. 5. The full length cDNA is 1.64 kb andencodes a mature CSF-1 protein of 224 amino acids. The clone wasdesignated CSF-17 with Cetus depository number CMCC 2347 and wasdeposited with the American Type Culture Collection on June 14, 1985, asaccession no. 53149. The plasmid bearing the CSF-1 encoding DNA wasdesignated pcCSF-17.

Mutein-Encoding Sequences

Modifications were made of the pcCSF-17 inserts to provide correspondingplasmids encoding muteins of the mCSF-1 protein. For site-specificmutagenesis, pcCSF-17 and M13mp18 were digested with the samerestriction enzyme excising the appropriate region of the CSF-1 codingsequence, and the excised sequence was ligated into the M13 vector.Second strand synthesis and recovery of the desired mutated DNA used thefollowing oligonucleotide primers:

for pro₅₂ CSF-1, 5'-TACCTTAAACCGGCATTTCTC-3', which creates a new HpaIIsite at codons 52-53;

for gln₅₂ CSF-1, 5'-TACCTTAAACAGGCCTTTCTC-3', which creates a new StuIsite at codons 52-53;

for asp₅₉ CSF-1, 5'-GGTACAAGATATCATGGAG-3', which creates a new EcoRVsite at codons 59-60.

After second strand extension using Klenow, the phage were transformedinto E. coli DG98 and the resulting plaques screened with kinasedlabeled probe. After plaque purification, the desired mutated insertswere returned to replace the unmutated inserts in pcCSF-1, yieldingpCSF-pro52, pCSF-gln52, and pCSF-asp59, respectively.

Plasmids containing three deletion mutants which encode ∇₁₅₈₋ CSF-1 werealso prepared: pCSF-Bam, pCSF-BamBcl, and pCSF-BamTGA. For pCSF-Bam,pcCSF-17 was digested with BamHI, and the upstream BamHI/BamHI fragmentof the coding region was isolated and religated to the vector fragment.The ligation mixture was transformed into E. coli MM294 and plasmidswith the correct orientation were isolated. The resulting pCSF-Bamencodes 158 amino acids of the CSF-1 protein fused to six residuesderived from the vector at the C-terminus: arg-his-asp-lys-ile-his.

For pCSF-BamBcl, which contains the entire CSF-1 encoding sequence,except that the serine at position 159 is mutated to a stop codon, thecoding sequence was excised from pcCSF-17 and ligated into M13 forsite-specific mutagenesis using the primer:5'-GAGGGATCCTGATCACCGCAGCTCC-3'. This results in a new BclI site atcodons 159-160. The mutated DNA was excised with BstXI/EcoRI and ligatedinto the BstXI/EcoRI digested pcCSF-17, the ligation mixture wastransformed into E. coli DG105, a dam⁻ host, and the plasmid DNAisolated.

For pCSF-BamTGA, in which the codons downstream of the 159-stop aredeleted, pCSF-BamBcl was digested with XhoI and BclI, and the insertligated into XhoI/BamHI digested pcCSF-17.

In addition, pCSF-Gly150, which contains a TGA stop codon instead ofhistidine at position 151, was prepared from the pcCSF-17 insert bysite-specific mutagenesis using the appropriate primer, as describedabove.

E.4. Transient Expression of CSF-1

Expression of pcCSF-17

The expression of plasmid DNA from CSF-17 (pcCSF-17) in COS-7 cells wasconfirmed and quantitated using the bone marrow proliferation assay, thecolony stimulation assay and the radioreceptor assay. It will berecalled that the specificity of the bone marrow proliferation assay forCSF-1 resides only in the ability of CSF-1 antiserum to diminishactivity; that for the colony stimulation assay, in the nature of thecolonies obtained. Both assays showed CSF-1 production to be of theorder of several thousand units per ml.

Bone Marrow Proliferation

For the bone marrow stimulation assay, which measures biologicalactivity of the protein, bone marrow cells from Balb/C mice were treatedwith serial dilutions of the 72 hour supernatants, and proliferation ofthe cells was measured by uptake of labeled thymidine, essentially asdescribed by Moore, R. N., et al, J Immunol (1983) 131:2374 andPrystowsky, M. B., et al, Am J Pathol (1984) 114:149. The medium frominduced MIAPaCa cells was used as control. Specificity for CSF-1 wasconfirmed by the ability of rabbit antisera raised against human urinaryCSF-1 to suppress thymidine uptake. The results for COS-7 cellsupernatants transfected with pcCSF-17 (CSF-17 supernatant) at a 1:16dilution are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                    .sup.3 H-thymidine incorporation                                              (cpm)                                                                         no      normal  antihuman                                                     add'ns  serum   CSF-1 serum                                       ______________________________________                                        medium         861       786    2682                                          MIAPaCa supernate                                                                           12255     16498   3302                                          CSF-17 supernate                                                                            16685     21996   2324                                          ______________________________________                                    

(The antihuman CSF-1 serum was prepared as described by Das, et al,supra.)

The MIAPaCa supernatant (at the 1:16 dilution used above) contained 125U/ml CSF activity corresponding to 2000 U/ml in the undilutedsupernatant, where 1 unit of colony stimulating activity is defined asthe amount of CSF needed to produce one colony from 10⁵ bone marrowcells/ml in the assay of Stanley, E. R., et al, J Lab Clin Med (1972)79:657.

These data show that the bone marrow stimulating activity is associatedwith CSF-1, since thymidine uptake is inhibited by anti-CSF-1 serum.Regression of results in this bone marrow proliferation assay obtainedat four dilutions ranging from 1:8 to 1:64 gave an estimated activityfor CSF-1 in CSF-17 supernatants of 2358 U/ml, which was diminished to424 U/ml in the presence of antiserum, but showed an apparent increaseto 3693 U/ml in the presence of non-immune serum. This was comparable tothe levels shown in the radioreceptor assay below.

Colony Stimulation

Direct assay of the CSF-17 supernatants for colony stimulation (Stanley,E. R., et al, J Lab Clin Med (supra)) showed 4287 U/ml, which wassubstantially unaffected by the presence of non-immune serum but reducedto 0 U/ml in the presence of rabbit antihuman CSF-1. This compares to2562 U/ml in the MIAPaCa supernatants. Eighty-five percent of thepcCSF-17 transformed COS-7 supernatant induced colonies had mononuclearmorphology; MIAPaCa supernatant induced colonies showed a 94%macrophage-6 granulocyte ratio.

Radioreceptor Assay

The radioreceptor assay measures competition between ¹²⁵ I-labeled CSF-1and the test compound for specific receptors on J774.2 mouse macrophagecells. MIAPaCa supernatant, assayed for colony stimulating activity asabove, was used as a standard (2000 U/ml). The CSF-1 concentration ofthe pcCSF-17 transformed COS-7 supernatant was found to be 2470 U/mlbased on a 1:10 dilution and 3239 U/ml based on a 1:5 dilution.

Thus, comparable values for CSF-1 concentration in the media of COS-7cells transformed with pcCSF-17 were found in all assays.

Expression of Muteins

In a similar manner to that described above for pcCSF-17, themutein-encoding plasmids were transfected into COS-A2 cells andtransient expression of CSF-1 activity was assayed by the bone marrowproliferation assay and by radioimmunoassay using anti-CSF antibodies.The expression product of pCSF-pro52 was inactive, indicating that, asexpected, substitution by proline is not conservative. All other muteinsshowed activity in both assays as shown by the results below:

    ______________________________________                                        Expression of CSF-1 Constructs in COS Cells                                            Radio-   Bone Marrow Assay (units/ml)                                CSF-1      immunoassay                                                                              Proliferation                                           Plasmid    (units/ml) (units/ml)   Colony                                     ______________________________________                                        pcCSF-17   3130       2798         11,100                                                3080       3487           9750                                                3540       3334         11,500                                     pCSF-pro52    54.8    <25           <100                                                    51.9    <25           <100                                                    45.3    <25           <100                                      pCSF-gln52 1890       2969           6200                                                2250       2308           5500                                                1910       2229           4400                                     pCSF-asp59 3210       3381           9000                                                4680       3417           6800                                                3470       2812         10,600                                     PCSF-Bam   9600       8048         22,600                                                8750       8441         21,900                                                8400       10,995       21,700                                     pCSF-BamBc1                                                                              8800                    26,000                                                10,700                  21,600                                                15,450                  24,200                                     pCSF-BamTGA                                                                              8450                    22,600                                                7550                    23,200                                                9700                    20,000                                     pCSF-Gly150                                                                              26,850                  55,710                                     ______________________________________                                    

E.5. Stable Expression of CSF-1

The COS-7 system provides recombinant CSF-1 by permitting replication ofand expression from the vector sequences. It is a transient expressionsystem.

The human CSF-1 sequence can also be stably expressed in procaryotic oreucaryotic systems. In general, procaryotic hosts offer ease ofproduction, while eucaryotes permit the use of the native signalsequence and carry out desired post-translational processing. This maybe especially important in the case of CSF-1 since the native protein isa dimer. Bacteria produce CSF-1 as a monomer, which would then besubjected to dimerizing conditions after extraction.

Procaryotic Expression

For procaryotic expression, the cDNA clone, or the genomic sequence withintrons excised by, for example, site-specific mutagenesis, is alteredto place an ATG start codon immediately upstream of the glutamic acid atthe N-terminus, and a HindIII site immediately upstream of the ATG inorder to provide a convenient site for insertion into the standard hostexpression vectors below. This can be done directly using insertionsite-specific mutagenesis with a synthetic oligomer containing a newsequence complementary to the desired AAGCTTATG, flanked by nucleotidesequences complementary to the native leader and N-terminal codingsequences.

For cDNA obtained using the method of Okayama and Berg, the DNA fragmentcontaining the entire coding sequence is excised from pcCSF-17 or thecorresponding mutein vector by digestion with XhoI (at sites retainedfrom the host cloning vector), isolated by agarose gel electrophoresis,and recovered by electroelution. To carry out the mutagenesis, the hostbacteriophage M13mp18 DNA is also treated with SalI and ligated with thepurified fragment under standard conditions and transfected into frozencompetent E. coli K12 strain DG98. The cells are plated on mediacontaining 5×10⁻⁴ M isopropyl thiogalactoside (IPTG) obtained from SigmaChem. (St. Louis, Mo.) and 40 μg/ml X-gal. Non-complementing whiteplaques are picked into fresh media. Mini-cultures are screened forrecombinant single strand phage DNA of the expected size, and thestructure of the desired recombinant phage is confirmed usingrestriction analysis.

A 34-mer complementary to the N-terminal and leader encoding portions ofthe CSF-1 sequence, but containing the complement to the desiredAAGCTTATG sequence, is synthesized and purified according to theprocedures set forth in C.4. A portion of this 34-mer preparation isradiolabeled according to a modification of the technique of Maxam andGilbert (Maxam, A., et al, Methods in Enzymology (1980) 68:521, AcademicPress) as set forth in C.4 above.

To perform the mutagenesis the above prepared recombinant bacteriophageis prepared in E. coli K12 strain DG98 and the single-strand phage DNApurified. One pmole of single-strand phage DNA and 10 pmoles of theabove synthetic nucleotide primer (not kinased) are annealed by heatingfor 1 min at 67° C., and then 30 min at 37° C. in 15 μl 20 mM Tris-Cl,pH 8, 20 mM MgCl₂, 100 mM NaCl, 20 mM 2-mercaptoethanol. The annealedDNA is incubated with DNA polymerase I (Klenow) and 500 μM dNTPs for 30min, 0° C. and then brought to 37° C. Aliquots (0.05 or 0.25 pmole) areremoved after 5 min, 20 min, and 45 min, transformed into E. coli K12strain DG98 and plated.

After growth, the plates are chilled at 4° C. and plaques lifted withPalI membranes obtained from Biodyne or S&S filters (1-2 min in thefirst filter, more than 10 min for the second filter). The filters aredenatured in 2.5M NaCl, 0.5M NaOH (5 min). The denaturing medium isneutralized with 3M sodium acetate to pH 5.5, or with 1M Tris-Cl, pH 7.5containing 1M NaCl, and the filters are baked at 80° C. in vacuo for 1hr, and then prehybridized at high stringency. The filters are thenprobed with the kinased synthetic 34-mer prepared above at highstringency, washed, and autoradiographed overnight at -70° C.

The RF form of the desired mutated phage is treated with EcoRI, bluntedwith Klenow, and then digested with HindIII to excise the gene as aHindIII/blunt fragment. (In a strictly analogous manner, the CSF-1encoding sequence from pMCSF may be obtained and modified.)

This fragment containing the human (or murine) CSF-1 encoding sequenceis then ligated with HindIII/BamHI (blunt) digested pPLOP or pTRP3 (seebelow) to place the coding sequence containing the ATG start codonimmediately downstream from the P_(L) or trp promoter respectively.These resulting plasmids are transformed into E. coli MC1000 lambdalysogen or MM294, and the cells grown under non-inducing conditions andthen induced by means appropriate to the promoter. The cells areharvested by centrifugation and sonicated and the liberated CSF-1 issolubilized. The presence of human (or murine) CSF-1 is confirmed bysubjecting the sonicate to the colony stimulating assay set forth above.

In addition, the plasmid pFC54.t (ATCC 39789) which contains the P_(L)promoter and the Bacillis thuringiensis positive retroregulatorysequence (as described in EPO Application Publication No. 717,331,published Mar. 29, 1985) was used as a host vector. pFC54.5 was digestedwith HindIII/BamHI(blunt), and the desired coding sequences are ligatedinto the vector using the HindIII/EcoRI(blunt) excised fragment frompcCSF-17 or the mutein encoding vectors described above. Aftertransformation into E. coli MC1000 lambda lysogen, and induction, CSF-1production was obtained and verified as described above.

Finally, it was possible to improve the level of CSF-1 production fromthe foregoing constructs by altering the third nucleotide in each of thefirst six codons of the N-terminus. pFC54.5 containing theCSF-1-encoding fragment was digested with HindIII/BstXI, and the excisedfragment (which contains the ATG and a short portion of the subsequentcoding sequence) was replaced by a synthetic HindIII/BstXI segmentwherein the first six codons have the sequence: GAAGAAGTTTCTGAATAT. Theresulting analogous expression vector represents no change in the aminoacid sequence encoded; however, the levels of expression are improvedwhen this modified vector is used.

Eucaryotic Expression

The Okayama-Berg plasmid pcCSF-17, containing the cDNA encoding humanCSF-1 under control of the SV40 promoter, can also be used to effectstable incorporation in monkey CV-1 cells, the parent cell line fromwhich the COS-7 line was derived. The corresponding vectors encoding themuteins as described above can also be used in an exactly analogous way.The host monkey CV-1 cells were grown to confluence and thencotransformed using 10 μg pcCSF-17 and various amounts (1, 2, 5 and 10μg) of pRSV-NEO2 (Gorman, C., et al, Science (1983) 221:551-553) per500,000 cells. The transformants were grown in DMEM with 10% FBS mediumcontaining 100 μg/ml of G418 antibiotic, to which the pRSV-NEO2 plasmidconfers resistance. The CV-1 cell line showed a G418 transformationfrequency of 10⁻⁵.12 colonies per 10⁶ cells per μg DNA.

The CV-1 cells were cotransformed as described above and selected inG418-containing medium. Resistant clones were tested for stability ofthe G418-resistant phenotype by growth in G418-free medium and thenreturned to G418-containing medium. The ability of these cultures tosurvive when returned to antibiotic-containing medium suggests that thepRSV-NEO2 DNA was integrated permanently into the cell genome. Sincecells stably transformed with a marker plasmid have about 50%probability of having integrated the DNA of a cotransfecting plasmid,about half of these cells will also contain pcCSF-17 DNA in theirchromosomal DNA.

Several clones of the G418-resistant pools of CV-1 cells which weredemonstrated to be stably transformed as above were picked and grown induplicate flasks to near confluence. One flask of each duplicate wasinfected with SV-40 virus at a multiplicity of infection of 5, and themedium was harvested 6 days after infection for assay for CSF-1 using aradioimmunoassay. The immunoassay is based on competition of ¹²⁵I-labeled MIAPaCa CSF-1 for "Rabbit 52" polyclonal antiserum raisedagainst purified human urinary CSF-1.

One of the selected CV-1 clones showed 2335 U/ml production of CSF-1,according to this assay, whereas cells not infected with SV-40 showedless than 20 U/ml. Controls using COS-7 cells transfected with 10 μgpcCSF-17 showed 2400 U/ml CSF-1 production without SV-40 infection.

The CSF-1 producing CV-1 cell line contains the pcCSF-17 DNA stablyintegrated into its genome, and thus can be used for production of CSF-1upon infection with SV-40. Infection is presumed to "rescue" thepcCSF-17 DNA from the genome, and provide the SV-40 T-antigen necessaryfor replication of the rescued DNA. Without SV-40 infection, theintegrated pcCSF-17 DNA is not effectively expressed.

Optimization of the expression of the CSF-1-encoding sequence by theCV-1 (CSF-17) cell line showed 6500-8000 U/ml when measured by theradioimmunoassay six days after SV-40 infection using a multiplicity ofinfection of at least 1, and a 10% FBS medium. Studies on expressionlevels at a multiplicity of 10 showed comparable production, butproduction was reduced upon removal of the FBS from the medium on thesecond day after infection. Adenovirus is a preferred infectious agentwhen used in conjunction with the CV-1 cell line.

In the alternative, appropriate control systems and host vectorspermitting expression in eucaryotic hosts have been used to receive theCSF-1-encoding inserts. For example, CHO cells and suitable vectors havebeen used, as described in U.S. Ser. No. 438,991, filed Nov. 1, 1982,now abandoned, assigned to the same assignee and incorporated herein byreference.

E.6. Activity of CSF-1

The activity of CSF-1 was determined using partially purified MIAPaCaCSF-1 or murine L cell CSF-1 as models for the CV-1-produced recombinantmaterial. CSF-1 was shown to enhance the production of interferon andtumor necrosis factor (TNF) by induced human monocytes by up to 10-fold.CSF-1 also was demonstrated to stimulate macrophage antitumor toxicity.

Stimulation of TNF Production by Human Monocytes

MIAPaCa CSF-1 was purified from the supernatant by calcium phosphate gelfiltration and lentil lectin chromatography. For assay of lymphokineproduction, peripheral blood-adherent cells were incubated in duplicateflasks containing 10⁷ cells each. One flask was treated with 1000 U/mlCSF-1 purified as above. After 3 days, the cells were harvested, andwashed, and resuspended at a cell concentration of 5×10⁵ /ml and platedin 24-well plates at 0.5 ml/well. The wells were treated with 10 μg/mlLPS and 20 ng/ml PMA for 48 hr and the supernatants were harvested forTNF assay. Cells treated with CSF showed TNF secretions approximatelynine-fold higher than the untreated cells (1500 U/ml, compared to 162U/ml).

Stimulation of Interferon Production by Human Monocytes

In an analogous experiment to determine the effect of CSF-1 oninterferon production, peripheral blood-adherent cells were incubatedfor 3 days in the presence and absence of 1000 U/ml CSF-1, as describedabove, harvested, resuspended at 5×10⁵ /ml, and plated in a 24-wellplate, as described above. The cells were induced for interferonproduction by addition of varying amounts of poly(I):poly(C). Thesupernatants were assayed for interferon production by their cytopathiceffect on VSV-infected GM 2504 cells. The CSF-1-stimulated cells showedproduction of 100 U/ml when induced with 50 μg/ml poly(I):poly(C),whereas comparably induced untreated cells produced less than 3 U/ml.

Stimulation of Myeloid CSF Production by Human Monocytes

Monocytes were incubated±CSF-1 for 3 days and then induced forproduction of myeloid CSF as in Table 2. The three representativeexperiments shown used blood from different donors.

                                      TABLE 2                                     __________________________________________________________________________    Myeloid CSF (U/ml)                                                                     Exp. 1     Exp. 2     Exp. 3                                         Induction                                                                              -CSF +CSF  -CSF +CSF  -CSF  +CSF                                     __________________________________________________________________________    medium   0    0     0    0     0     0                                        0.1 μg/ml LPS                                                                       --   --    0    0     0      80 ± 17                              1 μg/ml LPS                                                                         0    700 ± 72                                                                          40 ± 20                                                                        200 ± 20                                                                         103 ± 12                                                                         377 ± 57                              0.1 μg/ml LPS +                                                                     --   --    617 ± 50                                                                         993 ± 101                                                                       1120 ± 82                                                                        1280 ± 60                             2 ng/ml PMA                                                                   1 μg/ml LPS +                                                                       283 ± 42                                                                         983 ± 252                                                                       360 ± 92                                                                        1400 ± 180                                                                       537 ± 47                                                                         1080 ± 122                            2 ng/ml PMA                                                                   2 ng/ml PMA                                                                            --   370 ± 17                                                                         297 ± 6                                                                         183 ± 15                                                                         380 ± 52                                                                         716 ± 76                              __________________________________________________________________________

Therefore, CSF-1 stimulates myeloid CSF or colony stimulating activityproduction.

Stimulation of Tumor Cell Killing by Murine Macrophage; Comparison toother Colony Stimulating Factors

To assay macrophage stimulation, murine CSF-1 obtained fromL-cell-conditioned medium, was used as a model for the recombinantlyproduced CSF-1 from pcCSF-17 in an assay which showed stimulation of theability of murine macrophages to kill sarcoma targets. In this assay, 2hr adherent C3H/HeN mouse peritoneal macrophages were incubated for 1day in vitro with and without CSF-1 and then mixed at a 20:1 ratio with³ H-thymidine-labeled mouse sarcoma TU5 cells along with 10% v/vConA-induced (10 μg/ml) spleen lymphokine (LK), which contains gammainterferon. The release of labeled thymidine over the following 48 hrwas used as a measure of tumor cell killing. The effect of adding CSF-1as murine L-cell-conditioned medium containing 1200 U/ml CSF-1 is shownin the following table.

Purified murine CSF-1 and rhCSF-1 from CV-1 and E. coli (221) have alsobeen effective in this assay.

    ______________________________________                                        Treatment                 Increase Due                                        DAY     DAY            Kill   to CSF-1                                        0→1                                                                            1→3     %      %                                               ______________________________________                                        --      --             13                                                     --      LK             39                                                     --      CSF-1 + LK     49     26                                              CSF-1   LK             51     31                                              CSF-1   CSF-1 + LK     60     54                                              --      --              3                                                     --      LK             35                                                     --      CSF-1 + LK     47     34                                              CSF-1   --              7                                                     CSF-1   LK             49     40                                              CSF-I   CSF-1 + LK     69     97                                              ______________________________________                                    

Increase in the ability to kill the target cells was noted whether CSF-1was added during the preliminary 1 day of growth or during the period ofinduction; however, the most dramatic effects were observed when CSF-1was present during both of these periods.

The possibility of contaminating bacterial lipopolysaccharide (LPS) asthe cause of stimulation of monocytes and macrophages was excluded: TheLPS content of the applied CSF-1 was low (<0.3 ng/3000 U CSF-1, byLimulus amoebocyte lysate assay); activity was removed by application toan anti-CSF-1 column; polymyxin B was used to neutralize LPS; themacrophages from C3H/HeJ mice respond to CSF-1 but not to LPS.

CSF-GM was prepared from 6 mouse lungs obtained 5 hours after IVadministration of 5 μg LPS. The lungs were chopped and incubated for 3days in serum free medium, and the supernatant was depleted of CSF-1using a YYG106 affinity column (CSF-1 content reduced from 270 U/ml to78 U/ml). CSF-G was prepared from similarly treated LDI serum-freemedium. Both CSF-GM and CSF-G contents were assayed at 2000 U/ml bycolony stimulating assay.

The peritoneal macrophages were incubated with 40% of either of theforegoing media or with L-cell medium assayed at 2000 U/ml CSF-1 for 1day, and then incubated for 48 hours either with additional medium orwith LK, and assayed for TU5 killing as described above.

The results are shown in FIG. 6. While CSF-1 showed marked enhancementof toxicity to TU5, neither CSF-G nor CSF-GM had any effect.

In Vivo Test of CSF-1 for Anti-Tumor Efficacy

A total of 2×10⁷ units/mg of recombinantly produced CSF-1 from CV1 cellline (158) (LAL:2 ng/ml, 8 ng/ml) was injected intraperitoneally at 50μg/dose twice a day for five days into a 20 g mouse (3 mice per group)implanted subcutaneously with a Meth A sarcoma tumor 7 days earlier. Forsix days after the beginning of the CSF-1 treatment, the three untreatedand three treated mice were evaluated for body weights and tumorvolumes. On day 7, one mouse from each group was sacrificed forcomparative histopathological analysis (no gross signs). The fourremaining mice were evaluated for the usual 14-day period in the Meth Amodel.

There was no evidence of toxicity as measured by change in body weight.The results are provided in the table below:

    ______________________________________                                        Mean Change in Tumor Weight (ΔTW)                                       Day    CSF-1     Buffered Saline                                                                           % Treated/Cured                                  ______________________________________                                        3      3.0       2.2         91                                               6      2.6       6.8         38                                               7      4.1       8.0         51                                               8      5.7       11.0        52                                               14     13.9      29.4        47                                               ______________________________________                                         ΔTW = Ratio of the mean tumor volume at the day indicated to the        mean tumor volume at day 0 within a single group of mice.                

The results show that there was evidence for CSF-1-mediated efficacy,particularly at the day 6 tumor volume measurements. The differencesbetween the CSF-1 and control groups was greatest during a periodstarting several days after the commencement of treatment and severaldays thereafter, after which the tumor returned to its usual rate ofgrowth. These data suggest that multiple daily dosing (continuousinfusions to improve efficacy at this dose level, for longer periods oftime) or a higher dose level and altered schedule to include drugholiday may enhance efficacy.

Similar results were using CSF-1 (190) from E. coli and CSF-1 (221)∇₂₂₁-LCSF from E. coli.

In Vitro Test of CSF-1 Alone and with IFN-γ for Anti-Tumor Efficacy

Monocytes were isolated from normal human blood by spinning tubes forfive minutes at about 100 rpm to eliminate platelets. These were diluted1:1 with phosphate buffered saline (PBS) and layered 1:1 on Ficoll. Thenthe monocytes were centrifuged for 20 minutes at 2800 rpm, washed twotimes with PBS, and resuspended in Dulbecco's Modified Eagle's Mediumcontaining 5% adult bovine serum (ABS) (10 ml). Then the suspension waslayered 1:1 on 50% isotonic Percoll and centrifuged for 25 minutes at2800 rpm. The monocyte band was washed twice. A total of 7.1×10⁵cells/ml was counted. It was adjusted to 1.5 ml to 1.2×10⁶ cells/ml andplated in a 96-well plate at 100 μl/well and allowed to adhere for onehour at 37° C. Activators and media were added to bring the well volumesto 200 μl and the wells were incubated for 24 hours at 37° C. Theactivators were as follows:

1) media control

2) IFN-γ (100 units/ml) from Genzyme+LPS (10 μg/ml)

3) IFN-γ (1000 units/ml)+LPS (10 μg/ml)+rCSF-1 (mammalian CV-1-shortclone 158) (100 units/ml)

4) rCSF-1 (short clone 158) (1000 units/ml)

5) LPS-5 μg/ml.

Two T-25 flasks of A375 malignant human melanoma cell line target cellswere labeled with 0.5 μCi/ml of ¹²⁵ I-iododeoxyuridine in the same24-hour period. The target cells were harvested by trypsinization andcounted. The medium was aspirated from the monocytes, and 100 μl/well ofthe A375 cell line (10⁴ cells/well) was added for an effector:targetratio of 10:1. The plates were incubated (cells cocultured) for 72hours, and the wells washed one time with media. Then the wells werewashed twice and the remaining viable A375 cells were lysed by adding100 μl/well of 0.5N NaOH. The lysates were collected and counted in agamma counter and the % spontaneous and % generated cytotoxicity wascalculated as follows:

% spontaneous cytotoxicity:

    100-(A/C×100)

where A=cpm in cultures of control monocytes and target cells, and C=cpmin target cells alone.

% generated cytotoxicity

    100-(B/A×100)

where A=cpm in cultures of control monocytes and target cells, and B=cpmin cultures of treated monocytes and target cells.

The results are shown in the following table:

    ______________________________________                                                              Corrected % Generated                                   Activator(s)                                                                             Raw cpm    mean CPM  Cytotoxicity                                  ______________________________________                                        Media      957        951 ± 61                                             Control    1079                                                               IFN-γ and                                                                          819        539 ± 213                                                                            43                                            LPS        392                                                                rCSF-1     339        400 ± 127                                                                            60                                                       594                                                                A375       1902       1816 ± 19                                                       1863                                                               ______________________________________                                         % spontaneous cytotoxicity = 47%                                         

The results show that CSF-1 and LPS/IFN-γ exhibited significantcytotoxicity.

In Vitro Stimulation of Murine Antiviral Activity

Adherent murine thioglycolate-elicited macrophages were incubated withCSF-1 for 3 days and infected with VSV overnight. The following tableshows crystal violet staining of cells remaining adherent.

                  TABLE 3                                                         ______________________________________                                                           Absorbance                                                 Treatment          (mean)(S.D.)                                               ______________________________________                                        Medium/No virus    0.0346 ± 0.02                                           Medium             0.170 ± 0.02                                            CSF-1, 1000 U/ml + virus                                                                         0.264 ± 0.02                                            CSF-1, 2000 U/ml + virus                                                                         0.356 ± 0.04                                            LPS, 1 ng/ml       0.286 ± 0.03                                            ______________________________________                                    

CSF-1 treated cells, therefore, showed protection of the macrophageagainst VSV.

In Vitro Treatment of CMV Infection with CSF-1

Outbred CD1 mice were treated with the CSF-1 produced from the CV-1 cellline (short clone 158) in mammalian cells at doses of 400 μg/kg,intraperitoneally, once a day for five days, starting two days beforeinfection with a sub-lethal dose of cytomegalovirus (CMV). Mice weresacrificed on the third day after infection and the extent of viralreplication in target organs such as the spleen was evaluated by plaqueassay. The results showed that mice treated with CSF-1 havesignificantly lowered (57.8% reduction in) spleen viral titer comparedto the saline-treated control mice, indicating that CMV infection isless severe in CSF-1-treated mice.

Separately, CSF-1 produced in E. coli [N∇3∇₂₂₁ -CSF-1 (long clone 221)],has been tested in a lethal murine CMV infection model in outbred CD1mice (this is in contrast to the above experiment using sub-lethal dosesof CMV, in which organ titers were monitored). When CSF-1 wasadministered intraperitoneally to mice at 3-4 mg/kg (single dose givenper mouse) 24 hours before viral challenge, there was a significantincrease in survival as compared to saline-treated control.

Thus, CSF-1 may be used alone or in combination with another lymphokinein the treatment of viral infections in general, and in particular, maybe beneficial in immunosuppressive viral infection such as acquiredimmune deficiency syndrome (AIDS).

The preferred dosage range is about 350-450 μg CSF-1 per dose.

In Vivo Prophylactic Treatment of Bacterial Infection with CSF-1

Outbred CD1 mice were administered CSF-1, produced from the CV-1 cellline (short clone 158), intraperitoneally before challenge with a lethaldose of a clinical isolate of E. coli (SM18), a bacterium responsiblefor causing Gram-negative septis upon introduction into a host. The micewere then monitored for survival for 7 days post-infection.

Pretreatment with CSF-1 significantly enhanced survival of micechallenged with lethal doses of E. coli. The effect is dependent on thedose of CSF-1 and the schedule of administration.

In Vivo Stimulation of White Blood Cell Count

Outbred CD1 mice were administered purified recombinant human CSF-1, at2 mg/kg per dose, three times a day for five consecutive days. Totalwhite blood cell count increased to 12,000-13,000/μl in CSF-1-treatedmice from 8,700/μl in saline-treated control mice. In addition,neutrophil count increased to 6,821/μl in CSF-1-treated mice as comparedto 1,078/μl in saline-treated control mice.

This effect is dependent on the dose of CSF-1 and the schedule ofadministration. The increase in peripheral blood neutrophils wasdetectable 2-4 hours after a single dose of CSF-1 was administeredintraperitoneally. These results indicate that CSF-1 administration maybe useful in clinical or veterinary medicine as a stimulus ofgranulocyte and an enhancer of white blood count.

The three above experiments will also work with other forms of CSF-1 andmuteins thereof, for example, as described in U.S. Ser. Nos. 923,067 nowabandoned, 039,654 now abandoned, and 039,657, filed Oct. 24, 1986, Apr.16, 1987, and Apr. 16, 1987, respectively, the disclosures of all ofwhich are incorporated herein by reference.

The CSF-1 polypeptides may be produced in E. coli and other suitablehosts and recovered and refolded using the processes described in U.S.Ser. No. 040,174, filed Apr. 16, 1987 now U.S. Pat. No. 4,929,700.Alternatively, the CSF-1 polypeptides may be produced in baculovirushosts as disclosed in U.S. Ser. No. 077,188, filed July 24, 1987 nowabandoned. The disclosures of the above two applications areincorporated herein by reference.

E. 7. Formulation of CSF-1

The recombinantly produced human CSF-1 may be formulated foradministration using standard pharmaceutical procedures. OrdinarilyCSF-1 will be prepared in injectable form, and may be used either as thesole active ingredient, or in combination with other proteins or othercompounds having complementary or similar activity. Such other compoundsmay include alternate antitumor agents such as adriamycin, orlymphokines, such as IL-1, -2, -3, and -4, alpha-, beta-, andgamma-interferons, CSF-GM and CSF-G, and tumor necrosis factor. Theeffect of the CSF-1 active ingredient may be augmented or improved bythe presence of such additional components. As described above, theCSF-1 may interact in beneficial ways with appropriate blood cells, andthe compositions of the invention therefore include incubation mixturesof such cells with CSF-1, optionally in the presence of additionallymphokines. Either the supernatant fractions of such incubationmixtures, or the entire mixture containing the cells as well, may beused.

F. Murine CSF-1

An intronless DNA sequence encoding murine CSF-1 is prepared using amurine fibroblast cell line which produces large amounts of CSF-1. TheL-929 line, obtainable from ATCC, is used as a source for mRNA in orderto produce a cDNA library. Using oligomeric probes constructed on thebasis of the known murine N-terminal and CNBr-cleaved internal peptidesequence, this cDNA library is probed to retrieve the entire codingsequence for the murine form of the protein. Murine CSF-1 is believed tobe approximately 80% homologous to the human material because of thehomology of the N-terminal sequences, the ability of both human andmurine CSF-1 preparations to stimulate macrophage colonies from bonemarrow cells, and limited cross-reactivity with respect to radioreceptorand radioimmunoassays (Das, S. K., et al, Blood (1981) 58:630).

F.1. Protein Purification

Murine CSF-1 was purified by standard methods similar to those that aredisclosed by Stanley, E. R. et al, J Immunol Meth (1981) 42:253-284 andby Wang, F. F., et al, J Cell Biochem (1983) 21:263-275 or SDS gelelectrophoresis as reviewed by Hunkapiller, M. W., et al, Science (1984)226:304.

Amino acids 1-39 of the murine sequence were obtained, taking advantageof cyanogen bromide cleavage at position 10 to extend the degradationprocedure. An internal cleavage fragment from the mouse protein was alsoobtained and sequenced.

Overall composition data for the mouse protein were also obtained asshown below. These data show correct relative mole % for those aminoacids showing good recoveries; however the numbers are not absolute, ashistidine and cysteine were not recovered in good yield.

    ______________________________________                                        Amino Acid     mole %   residues/125                                          ______________________________________                                        Asp            20.1     25.1                                                  Glu            20.0     25.0                                                  His            --       --                                                    Ser            6.0      7.5                                                   Thr            5.9      7.4                                                   Gly            5.4      6.8                                                   Ala            6.8      8.5                                                   Arg            3.0      3.8                                                   Pro            6.7      8.4                                                   Val            5.3      6.6                                                   Met            1.1      1.4                                                   Ile            3.9      4.9                                                   Leu            8.5      10.6                                                  Phe            6.0      7.5                                                   Lys            3.5      4.4                                                   Tyr            4.1      5.1                                                   ______________________________________                                    

The conversion to residues/125 was based on an approximation of sequencelength from molecular weight.

F.2. Preparation of Murine CSF-1 cDNA

The amino acid sequence 5-13 of the murine CSF-1 and the internalsequence were used as a basis for probe construction.

Three sets of oligomers corresponding to the murine sequence wereprepared. One sequence was prepared to encode "region A"--i.e., aminoacids 9-13; another was prepared to "region B"--i.e., amino acids 5-9,as shown in FIG. 2; a third to encode positions 0-6 of an internalsequence, "region C". Because of codon redundancy, each of these classesof oligomers is highly degenerate.

Thus, 15-mers constructed on the basis of region A number 48; 14-mersconstructed on the basis of region B (deleting the last nucleotide ofthe codon for histidine) also number 48; 20-mers constructed on thebasis of region C number 32. Alternatively stated, a 15-mer constructedso as to encode region A may have a mismatch in four of the fifteenpositions; a particular 14-mer constructed with respect to region B mayhave a mismatch in six positions; a particular 20-mer constructed withrespect to region C may have a mismatch in five positions.

As described below, by suitable protocol design, an enriched messengerRNA fraction may be found for the production of the desired enrichedmurine cDNA library, and the precisely correct oligomers for use asprobes also ascertained.

Total messenger RNA is extracted and purified from murine L-929 cells.Murine L-929 cells are cultured for 8 days on DME medium and thenharvested by centrifugation. Total cytoplasmic ribonucleic acid (RNA)was isolated from the cells by the same protocol as set forth above forMIAPaCa mRNA.

The mRNA is fractionated on gels for Northern blot as described inparagraph C.3. The 15-mer sequences corresponding to region A aredivided into four groups of twelve each. Each of these groups was usedto hydridize under low stringency both to control and to murine L-929mRNA slabs and the resulting patterns were viewed by radioautography.Under the low stringency conditions employed, hybridization occurs tofractions not containing the proper sequence, as well as to those thatdo. Also, because the control cell line is different from that of theL-929 line in ways other than failure to produce CSF-1, hybridizationoccurs in a number of size locations not related to CSF-1 in the L-929cell gels which are not present in the controls.

Comparable sets of control and L-929 gels are probed with segregants ofthe 48 14-mers representing region B and segregants of the 32 20-mersrepresenting region C. Only the bands of messenger RNA which hybridizeexclusively in the L-929 slabs for either regions A or B, and C probesare then further considered.

The RNA band which continues to bind to one of the A region 15-mermixture or one of the region B 14-mer mixture and one of the region C20-mer mixture under conditions of increasingly higher stringency isselected.

When the correct mRNA band is found, each of the groups of region A15-mers is used to probe at various stringency conditions. The groupbinding at highest stringency presumably contains the correct 15-merexactly to complement the mRNA produced. The correct 15-mer isascertained by further splitting the preparation until a single oligomeris found which binds at the highest stringency. A similar approach isused to ascertain the correct 14-mer or 20-mer which binds to region Bor C. These specific oligomers are then available as probes in a murinecDNA library which is prepared from the enriched mRNA fraction.

The mRNA fraction identified as containing the coding sequence for CSF-1is then obtained on a preparative scale. In this preparation, the polyA⁺ mRNA was fractionated on a sucrose gradient in 10 mM Tris-HCl, pH7.4, 1 mM EDTA, and 0.5% SDS. After centrifugation in a Beckman SW40rotor at 30,000 rpm for 17 hr, mRNA fractions are recovered from thegradient by ethanol precipitation. RNA fractions recovered from thegradient were each injected into Xenopus oocytes in a standardtranslation assay and the products assayed for CSF-1 usingradioimmunoassay with antibodies raised against murine CSF-1. Fractionsfor which positive results were obtained were pooled and used toconstruct the cDNA library. These same fractions hybridize to theoligomeric probes.

Other methods of preparing cDNA libraries are, of course, well known inthe art. One, now classical, method uses oligo dT primer, reversetranscriptase, tailing of the double-stranded cDNA with poly dG, andannealing into a suitable vector, such as pBR322 or a derivativethereof, which has been cleaved at the desired restriction site andtailed with poly dC. A detailed description of this alternative methodis found, for example, in U.S. Pat. No. 4,518,584, issued May 21, 1985,incorporated herein by reference.

In the method used here, the enriched mRNA (5 μg) is denatured bytreatment with 10 mM methyl mercury at 22° C. for 5 min and detoxifiedby the addition of 100 mM 2-mercaptoethanol (Payvar, F., et al, J BiolChem (1979) 254:7636-7642). Plasmid pcDV1 is cleaved with KpnI, tailedwith dTTP, and annealed to the denatured mRNA. This oligo dT primed mRNAis treated with reverse transcriptase, and the newly synthesized DNAstrand tailed with dCTP. Finally, the unwanted portion of the pcDV1vector is removed by cleavage with HindIII. Separately, pL1 is cleavedwith PstI, tailed with dGTP, cleaved with HindIII, and then mixed withthe poly T tailed mRNA/cDNA complex extended by the pcDV1 vectorfragment, and ligated with E. coli ligase, and the mixture treated withDNA polymerase I (Klenow) E. coli ligase, and RNase H. The resultingvectors are transformed into E. coli K12 MM294 to Amp^(R).

The resulting cDNA library is then screened using the oligomer probesidentified as complementary to the mRNA coding sequence as describedabove. Colonies hybridizing to probes from regions A or B and C arepicked and grown; plasmid DNA is isolated, and plasmids containinginserts of sufficient size to encode the entire sequence of CSF-1 areisolated. The sequence of the insert of each of these plasmids isdetermined, and a plasmid preparation containing the entire codingsequence including regions A and B at the upstream portion is designatedpcMCSF.

F.3 Expression of Murine CSF-1 DNA

In a manner similar to that set forth above for the human cDNA, themurine cDNA is tested for transient expression in COS cells, and usedfor expression in stably transformed CV-1. In addition, the appropriateHindIII/ATG encoding sequences are inserted upstream of the matureprotein by mutagenesis and the coding sequences inserted into pPLOP orpTRP3 for procaryotic expression.

G. Host Vectors

pPLOP is a host expression vector having the P_(L) promoter and N generibosome binding site adjacent a HindIII restriction cleavage site, thuspermitting convenient insertion of a coding sequence having an ATG startcodon preceded by a HindIII site. The backbone of this vector is atemperature-sensitive high copy number plasmid derived from pCS3. pPLOPwas deposited at ATCC on Dec. 18, 1984, and has accession number 39947.

pTRP3 is a host expression vector containing a trp promoter immediatelyupstream of a HindIII restriction site, thus permitting insertion of acoding sequence in a manner analogous to that above for pPLOP. Thebackbone vector for pTRP3 is pBR322. pTRP3 was deposited with ATCC onDec. 18, 1984, and has accession number 39946.

Construction of pPLOP

Origin of Replication

pCS3 provides an origin of replication which confers high copy number ofthe pPLOP host vector at high temperatures. Its construction isdescribed extensively in copending U.S. Ser. No. 541,948, filed Oct. 14,1983, incorporated herein by reference. pCS3 was deposited June 3, 1982and assigned ATCC number 39142.

pCS3 is derived from pEW27 and pOP9. pEW27 is described by E. M. Wong,Proc Natl Acad Sci (U.S.A.) (1982) 79:3570. It contains mutations nearits origin of replication which provide for temperature regulation ofcopy number. As a result of these mutations replication occurs in highcopy number at high temperatures, but at low copy number at lowertemperatures.

pOP9 is a high copy number plasmid at all temperatures which wasconstructed by inserting into pBR322 the EcoRI/PvuII origin containingfragment from Co1 E1 type plasmid pOP6 (Gelfand, D., et al, Proc NatlAcad Sci (U.S.A.) (1978) 75:5869). Before insertion, this fragment wasmodified as follows: 50 μg of pOP6 was digested to completion with 20units each BamHI and SstI. In order to eliminate the SstI 3' protrudingends and "fill in" the BamHI 5' ends, the digested pOP6 DNA was treatedwith E. coli DNA polymerase I (Klenow in a two-stage reaction first at20° C. for elimination of the 3' SstI protruding end and then at 9° C.for repair at the 5' end. The blunt-ended fragment was digested and 0.02pmole used to transform competent DG75 (O'Farrell, P., et al, JBacteriology (1978) 134:645-654). Transformants were selected on Lplates containing 50 μ/ml ampicillin and screened for a 3.3 kb deletion,loss of an SstI site, and presence of a newly formed BamHI site.

One candidate, designated pOP7, was chosen and the BamHI site deleted bydigesting 25 μg of pOP7 with 20 units BamHI, repairing with E. coli DNApolymerase I fragment (Klenow), and religating with T4 DNA ligase.Competent DG75 was treated with 0.1 μg of the DNA and transformants wereselected on L plates containing 50 μg/ml ampicillin. Candidates werescreened for the loss of the BamHI restriction site. pOP8 was selected.To obtain pOP9 the AvaI (repaired)/EcoRI Tet^(R) fragment from pBR322was prepared and isolated and ligated to the isolated PvuII(partial)/EcoRI 3560 bp fragment from pOP8.

Ligation of 1.42 kb EcoRI/AvaI (repair) Tet^(R) (fragment A) and 3.56 kbEcoRI/PvuII Amp^(R) (fragment B) used 0.5 μg of fragment B and 4.5 μg offragment A in a two-stage reaction in order to favor intermolecularligation of the EcoRI ends.

Competent DG75 was transformed with 5 μl of the ligation mixture, andtransformants were selected on ampicillin (50 μg/ml)-containing plates.pOP9, isolated from Amp^(R) Tet^(R) transformants, showed high copynumber, colicin resistance, single restriction sites for EcoRI, BamHI,PvuII, HindIII, 2 restriction sites for HincII, and the appropriate sizeand HaeIII digestion pattern.

To obtain pCS3, 50 μg pEW27 DNA was digested to completion with PvuIIand the EcoRI. Similarly, 50 μg of pOP9 was digested to completion withPvuII and EcoRI and the 3.3 kb fragment was isolated.

0.36 μg (0.327 pmoles) pEW27 fragment and 0.35 μg (0.16 pmoles) pOP9fragment were ligated and used to transform E. coli MM294. Amp^(R)Tet^(R) transformants were selected. Successful colonies were initiallyscreened at 30° C. and 41° C. on beta-lactamase assay plate and then forplasmid DNA levels following growth at 30° C. and 41° C. A successfulcandidate, designated pCS3, was confirmed by sequencing.

Preparation of the P_(L) N_(RBS) Insert

The DNA sequence containing P_(L) phage promoter and the ribosomebinding site for the N-gene (N_(RBS)) was obtained from pFC5, andultimately from a derivative of pKC30 described by Shimatake andRosenberg, Nature (1981) 292:128. pKC30 contains a 2.34 kb fragment fromlambda phage cloned into the HindIII/BamHI vector fragment from pBR322.The P_(L) promoter and N_(RBS) occupy a segment in pKC30 between a BgIIIand HpaI site. The derivative of pKC30 has the Bg1II site converted toan EcoRI site.

The Bg1II site immediately preceding the P_(L) promoter was convertedinto an EcoRI site as follows: pKC30 was digested with Bg1II, repairedwith Klenow and dNTPs and ligated with T4 ligase to an EcoRI linker(available from New England Biolabs) and transformed into E. coli K12strain MM294 lambda⁺. Plasmids were isolated from Amp^(R) Tet^(R)transformants and the desired sequence was confirmed by restrictionanalysis and sequencing. The resulting plasmid, pFC3was double-digestedwith PvuI and HpaI to obtain an approximately 540-bp fragment isolatedand treated with Klenow and dATP, followed by S1 nuclease, to generate ablunt-ended fragment with the 3' terminal sequence -AGGAGAA where the-AGGAGA portion is the N_(RBS). This fragment was restricted with EcoRIto give a 347 base pair DNA fragment with 5'-EcoRI (sticky) and HinfI(partial repair, S1 blunt)-3' termini.

To complete pFC5, pβI-Z15 was used to create a HindIII site 3' of theN_(RBS). pβI-Z15 was deposited Jan. 13, 1984. ATCC No. 39578, and wasprepared by fusing a sequence containing ATG plus 140 bp of β-IFN fusedto lac Z into pBR322. In pβI-Z15, the EcoRI site of pBR322 is retained,and the insert contains a HindIII site immediately preceding the ATGstart codon of β-IFN. pβI-Z15 was restricted with HindIII, repaired withKlenow and dNTPs, and then digested with EcoRI. The resultingEcoRI/HindIII (repaired) vector fragment was ligated with theEcoRI/HinfI (repaired) fragment above, and the ligation mixture used totransform MC1000-39531. Transformants containing the successfulconstruction were identified by ability to grow on lactose minimalplates at 34° C. but not at 30° C. (Transformations were plated onX-gal-Amp plates at 30° C. and 34° C. and minimal-lactose plates at 30°C. and 34° C. Transformants with the proper construction are blue onX-gal-Amp plates at both temperatures, but on minimal lactose plates,grow only at 34° C.) The successful construct was designated pFC5.

Completion of pPLOP

pCS3 was then modified to provide the P_(L) and N_(RBS) controlsequences. pCS3 was digested with HindIII, and then digested with EcoRI.The vector fragment was ligated with an isolated EcoRI/HindIII from pFC5containing the P_(L) N_(RBS) and transformed into E. coli MM294. Thecorrect construction of isolated plasmid DNA was confirmed byrestriction analysis and sequencing and the plasmid designated pPLOP.

Preparation of pTRP3

To construct the host vector containing the trp control sequences behinda HindIII site, the trp promoter/operator/ribosome binding sitesequence, lacking the attenuator region, was obtained from pVH153,obtained from C. Yanofsky, Stanford University. Trp sequences areavailable in a variety of such plasmids known in the art. pVH153 wastreated with HhaI (which cuts leaving an exposed 3' sticky end just 5'of the trp promoter) blunt ended with Klenow, and partially digestedwith TaqI. The 99 bp fragment corresponding to restriction at the TaqIsite, 6 nucleotides preceding the ATG start codon of trp leader, wereisolated, and then ligated to EcoRI (repair)/ClaI digested, pBR322 toprovide pTRP3.

On Apr. 2, 1985, Applicants have deposited with the American TypeCulture Collection, Rockville, Md., U.S.A. (ATCC) the phage pHCSF-1 inE. coli DG98, accession no. 40177. On May 21, 1985, pHCSF-1a, designatedCMCC 2312 in the Cetus collection and pHCSF-1 λ Charon 4A for deposit,was deposited with ATCC and has accession no. 40185. On June 14, 1985,CSF-17 in E. coli MM294, designated CMCC 2347, was deposited with ATCCand has accession no. 53149. In addition, the following deposits weremade with ATCC on the date of June 19, 1986:

    ______________________________________                                        Plasmid         CMCC No.  ATCC No.                                            ______________________________________                                        pCSF-asp59      2705      67139                                               PCSF-gln52      2708      67140                                               pCSF-pro52      2709      67141                                               pCSF-Bam        2710      67142                                               PCSF-BamBc1     2712      67144                                               PCSF-Gly150     2762      67145                                               ______________________________________                                    

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromdate of deposit. The deposits will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenApplicants and ATCC which assures permanent and unrestrictedavailability upon issuance of the pertinent U.S. patent. The Assigneeherein agrees that if the culture on deposit should die or be lost ordestroyed when cultivated under suitable conditions, it will be promptlyreplaced upon notification with a viable specimen of the same culture.Availability of the deposits is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

These deposits were made for the convenience of the relevant public anddo not constitute an admission that a written description would not besufficient to permit practice of the invention or an intention to limitthe invention to these specific constructs. Set forth hereinabove is acomplete written description enabling a practitioner of ordinary skillto duplicate the constructs deposited and to construct alternative formsof DNA, or organisms containing it, which permit practice of theinvention as claimed.

The scope of the invention is not to be construed as limited by theillustrative embodiments set forth herein, but is to be determined inaccordance with the appended claims.

We claim:
 1. A method to enhance the production of interferon frommonocytes which comprises treating said monocytes with an effectiveamount of recombinant colony stimulating factor-1 (CSF-1).
 2. A methodto enhance the production of tumor necrosis factor (TNF) from monocyteswhich comprises treating said monocytes with an effective amount ofrecombinant colony stimulating factor-1 (CSF-1).
 3. A method to induceresistance to viral infections in macrophages which comprises treatingsaid macrophages with an effective amount of recombinant colonystimulating factor (CSF-1).
 4. A method in accordance with claim 3 toinduce resistance to CMV which comprises treating macrophages with aneffective amount of recombinant colony stimulating factor (CSF-1).
 5. Amethod to enhance killing of sarcoma tumor cells comprising treatingmonocytes or macrophages with an effective amount of human recombinantcolony stimulating factor-1 (CSF-1).